Cocoa extract compounds and methods for making and using the same

ABSTRACT

Polyphenol-containing compositions, for example cocoa procyanidin monomer and/or oligomer-containing compositions, and their use for inhibiting bacterial growth are disclosed. Compositions may be used for human and veterinary animal administration and may be, for example, in a form of a food, a dietary supplement, or a pharmaceutical.

REFERENCE TO RELATED APPLICATION

[0001] Reference is made to copending U.S. application Ser. Nos.08/709,406, filed Sep. 6, 1996, 08/631,661, filed Apr. 2, 1996, and08/317,226, filed Oct. 3, 1994 (now U.S. Pat. No. 5,554,645) andPCT/US96/04497, each of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to cocoa extracts and compounds therefromsuch as polyphenols preferably polyphenols enriched with procyanidins.This invention also relates to methods for preparing such extracts andcompounds, as well as to uses for them; for instance, as antineoplasticagents, anti oxidants, DNA topoisomerase II enzyme inhibitors,cyclo-oxygenase and/or lipoxygenase modulators, NO (Nitric Oxide) orNO-synthase modulators, as non-steroidal antiinflammatory agents,apoptosis modulators, platelet aggregation modulators, blood or in vivoglucose modulators, antimicrobials, and inhibitors of oxidative DNAdamage.

[0003] Documents are cited in this disclosure with a full citation foreach appearing thereat or in a References section at the end of thespecification, preceding the claims. These documents pertain to thefield of this invention; and, each document cited herein is herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

[0004] Polyphenols are an incredibly diverse group of compounds(Ferreira et al., 1992) which widely occur in a variety of plants, someof which enter into the food chain. In some cases they represent animportant class of compounds for the human diet. Although some of thepolyphenols are considered to be nonnutrative, interest in thesecompounds has arisen because of their possible beneficial effects onhealth.

[0005] For instance, quercetin (a flavonoid) has been shown to possessanticarcinogenic activity in experimental animal studies (Deshner etal., 1991 and Kato et al., 1983). (+)-Catechin and (−)-epicatechin(flavan-3-ols) have been shown to inhibit Leukemia virus reversetranscriptase activity (Chu et al., 1992). Nobotanin (an oligomerichydrolyzable tannin) has also been shown to possess anti-tumor activity(Okuda et al., 1992). Statistical reports have also shown that stomachcancer mortality is significantly lower in the tea producing districtsof Japan. Epigallocatechin gallate has been reported to be thepharmacologically active material in green tea that inhibits mouse skintumors (Okuda et al., 1992). Ellagic acid has also been shown to possessanticarcinogen activity in various animal tumor models (Bukharta et al.,1992). Lastly, proanthocyanidin oligomers have been patented by theKikkoman Corporation for use as antimutagens. Indeed, the area ofphenolic compounds in foods and their modulation of tumor development inexperimental animal models has been recently presented at the 202ndNational Meeting of The American Chemical Society (Ho et al., 1992;Huang et al., 1992).

[0006] However, none of these reports teaches or suggests cocoa extractsor compounds therefrom, any methods for preparing such extracts orcompounds therefrom, or, any uses for cocoa extracts or compoundstherefrom, as antineoplastic agents, antioxidants, DNA topoisomerase IIenzyme inhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO(Nitric Oxide) or NO-synthase modulators, as non-steroidalantiinflammatory agents, apoptosis modulators, platelet aggregationmodulators, blood or in vivo glucose modulators, antimicrobials, orinhibitors of oxidative DNA damage.

OBJECTS AND SUMMARY OF THE INVENTION

[0007] Since unfermented cocoa beans contain substantial levels ofpolyphenols, the present inventors considered it possible that similaractivities of and uses for cocoa extracts, e.g., compounds within cocoa,could be revealed by extracting such compounds from cocoa and screeningthe extracts for activity. The National Cancer Institute has screenedvarious Theobroma and Herrania species for anti-cancer activity as partof their massive natural product selection program. Low levels ofactivity were reported in some extracts of cocoa tissues, and the workwas not pursued. Thus, in the antineoplastic or anti-cancer art, cocoaand its extracts were not deemed to be useful; i.e., the teachings inthe antineoplastic or anti-cancer art lead the skilled artisan away fromemploying cocoa and its extracts as cancer therapy.

[0008] Since a number of analytical procedures were developed to studythe contributions of cocoa polyphenols to flavor development (Clappertonet al., 1992), the present inventors decided to apply analogous methodsto prepare samples for anti-cancer screening, contrary to the knowledgein the antineoplastic or anti-cancer art. Surprisingly, and contrary tothe knowledge in the art, e.g., the National Cancer Institute screening,the present inventors discovered that cocoa polyphenol extracts whichcontain procyanidins, have significant utility as anti-cancer orantineoplastic agents.

[0009] Additionally, the inventors demonstrate that cocoa extractscontaining procyanidins and compounds from cocoa extracts have utilityas antineoplastic agents, antioxidants, DNA topoisomerase II enzymeinhibitors, cyclo-oxygenase and/or lipoxygenase modulators, NO (NitricOxide) or NO-synthase modulators, as non-steroidal antiinflammatoryagents, apoptosis modulators, platelet aggregation modulators, blood orin vivo glucose modulators, antimicrobials, and inhibitors of oxidativeDNA damage.

[0010] It is an object of the present invention to provide a method forproducing cocoa extract and/or compounds therefrom.

[0011] It is another object of the invention to provide a cocoa extractand/or compounds therefrom.

[0012] It is still another object of the present invention to provide apolymeric compound of the formula A_(n), wherein A is a monomer havingthe formula:

[0013] wherein

[0014] n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and a plurality of additional monomericunits;

[0015] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0016] bonding between adjacent monomers takes place at positions 4, 6or 8;

[0017] a bond of an additional monomeric unit in position 4 has α or βstereochemistry;

[0018] X, Y and Z are selected from the group consisting of monomericunit A, hydrogen, and a sugar, with the provisos that as to the at leastone terminal monomeric unit, bonding of the additional monomeric unitthereto is at position 4 and Y=Z=hydrogen;

[0019] the sugar is optionally substituted with a phenolic moiety at anyposition, for instance, via an ester bond,

[0020] and pharmaceutically acceptable salts or derivatives thereof(including oxidation products).

[0021] It is still a further object of the present invention to providea polymeric compound of the formula A_(n), wherein A is a monomer havingthe formula:

[0022] wherein

[0023] n is an integer from 2 to 18, e.g., 3 to 18;

[0024] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0025] adjacent monomers bind at position 4 by (4→6) or (4→8);

[0026] each of X, Y and Z is H, a sugar or an adjacent monomer, with theprovisos that if X and Y are adjacent monomers, Z is H or sugar and if Xand Z are adjacent monomers, Y is H or sugar, and that as to at leastone of the two terminal monomers, bonding of the adjacent monomer is atposition 4 and optionally, Y=Z=hydrogen;

[0027] a bond at position 4 has α or β stereochemistry;

[0028] the sugar is optionally substituted with a phenolic moiety at anyposition, for instance, via an ester bond,

[0029] and pharmaceutically acceptable salts or derivatives thereof(including oxidation products).

[0030] It is another object of the invention to provide an antioxidantcomposition.

[0031] It is another object of the invention to demonstrate inhibitionof DNA topoisomerase II enzyme activity.

[0032] It is yet another object of the present invention to provide amethod for treating tumors or cancer.

[0033] It is still another object of the invention to provide ananti-cancer, anti-tumor or antineoplastic compositions.

[0034] It is still a further object of the invention to provide anantimicrobial composition.

[0035] It is yet another object of the invention to provide acyclo-oxygenase and/or lipoxygenase modulating composition.

[0036] It is still another object of the invention to provide an NO orNO-synthase-modulating composition.

[0037] It is a further object of the invention to provide anon-steroidal antiinflammatory composition.

[0038] It is another object of the invention to provide a blood or invivo glucose-modulating composition.

[0039] It is yet a further object of the invention to provide a methodfor treating a patient with an antineoplastic, antioxidant,antimicrobial, cyclo-oxygenase and/or lipoxygenase modulating or NO orNO-synthase modulating non-steroidal antiinflammatory modulating and/orblood or in vivo glucose-modulating composition.

[0040] It is an additional object of the invention to providecompositions and methods for inhibiting oxidative DNA damage.

[0041] It is yet an additional object of the invention to providecompositions and methods for platelet aggregation modulation.

[0042] It is still a further object of the invention to providecompositions and methods for apoptosis modulation.

[0043] It is a further object of the invention to provide a method formaking any of the aforementioned compositions.

[0044] And, it is an object of the invention to provide a kit for use inthe aforementioned methods or for preparing the aforementionedcompositions.

[0045] It has been surprisingly discovered that cocoa extract, andcompounds therefrom, have anti-tumor, anti-cancer or antineoplasticactivity or, is an antioxidant composition or, inhibits DNAtopoisomerase II enzyme activity or, is an antimicrobial or, is acyclo-oxygenase and/or lipoxygenase modulator or, is a NO or NO-synthasemodulator, is a non-steroidal antiinflammatory agent, apoptosismodulator, platelet aggregation modulator or, is a blood or in vivoglucose modulator, or is an inhibitor of oxidative DNA damage.

[0046] Accordingly, the present invention provides a substantially purecocoa extract and compounds therefrom. The extract or compoundspreferably comprises polyphenol(s) such as polyphenol(s) enriched withcocoa procyanidin(s), such as polyphenols of at least one cocoaprocyanidin selected from (−) epicatechin, (+) catechin, procyanidinB-2, procyanidin oligomers 2 through 18, e.g., 3 through 18, such as 2through 12 or 3 through 12, preferably 2 through 5 or 4 through 12, morepreferably 3 through 12, and most preferably 5 through 12, procyanidinB-5, procyanidin A-2 and procyanidin C-1.

[0047] The present invention also provides an anti-tumor, anti-cancer orantineoplastic or antioxidant or DNA topoisomerase II inhibitor, orantimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, or anNO or NO-synthase modulator, nonsteroidal antiinflammatory agent,apoptosis modulator, platelet aggregation modulator, blood or in vivoglucose modulator, or oxidative DNA damage inhibitory compositioncomprising a substantially pure cocoa extract or compound therefrom orsynthetic cocoa polyphenol(s) such as polyphenol(s) enriched withprocyanidin(s) and a suitable carrier, e.g., a pharmaceutically,veterinary or food science acceptable carrier. The extract or compoundtherefrom preferably comprises cocoa procyanidin(s). The cocoa extractor compounds therefrom is preferably obtained by a process comprisingreducing cocoa beans to powder, defatting the powder and, extracting andpurifying active compound(s) from the powder.

[0048] The present invention further comprehends a method for treating apatient in need of treatment with an anti-tumor, anti-cancer, orantineoplastic agent or an antioxidant, or a DNA topoisomerase IIinhibitor, or antimicrobial, or cyclo-oxygenase and/or lipoxygenasemodulator, or an NO or NO-synthase modulator, non-steroidalantiinflammatory agent, apoptosis modulator, platelet aggregationmodulator, blood or in vivo glucose modulator or inhibitor of oxidativeDNA damage, comprising administering to the patient a compositioncomprising an effective quantity of a substantially pure cocoa extractor compound therefrom or synthetic cocoa polyphenol(s) or procyanidin(s)and a carrier, e.g., a pharmaceutically, veterinary or food scienceacceptable carrier. The cocoa extract or compound therefrom can be cocoaprocyanidin(s); and, is preferably obtained by reducing cocoa beans topowder, defatting the powder and, extracting and purifying activecompound(s) from the powder.

[0049] Additionally, the present invention provides a kit for treating apatient in need of treatment with an anti-tumor, anti-cancer, orantineoplastic agent or antioxidant or DNA topoisomerase II inhibitor,or antimicrobial, or cyclo-oxygenase and/or lipoxygenase modulator, oran NO or NO-synthase modulator, non-steroidal antiinflammatory agent,apoptosis modulator, platelet aggregation modulator inhibitor ofoxidative DNA damage, or blood or in vivo glucose modulator comprising asubstantially pure cocoa extract or compounds therefrom or syntheticcocoa polyphenol(s) or procyanidin(s) and a suitable carrier, e.g., apharmaceutically, veterinary or food science acceptable carrier, foradmixture with the extract or compound therefrom or syntheticpolyphenol(s) or procyanidin(s).

[0050] The present invention provides compounds as illustrated in FIGS.38A to 38P and 39A to 39AA; and linkages of 4→6 and 4→8 are presentlypreferred.

[0051] The invention even further encompasses food preservation orpreparation compositions comprising an inventive compound, and methodsfor preparing or preserving food by adding the composition to food.

[0052] And, the invention still further encompasses a DNA topoisomeraseII inhibitor comprising an inventive compound and a suitable carrier ordiluent, and methods for treating a patient in need of such treatment byadministration of the composition.

[0053] Considering broadly the aforementioned embodiments involvingcocoa extracts, the invention also includes such embodiments wherein aninventive compound is used instead of or as the cocoa extracts. Thus,the invention comprehends kits, methods, and compositions analogous tothose above-stated with regard to cocoa extracts and with an inventivecompound.

[0054] These and other objects and embodiments are disclosed or will beobvious from the following Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The following Detailed Description will be better understood byreference to the accompanying drawings wherein:

[0056]FIG. 1 shows a representative gel permeation chromatogram from thefractionation of crude cocoa procyanidins;

[0057]FIG. 2A shows a representative reverse-phase HPLC chromatogramshowing the separation (elution profile) of cocoa procyanidins extractedfrom unfermented cocoa;

[0058]FIG. 2B shows a representative normal phase HPLC separation ofcocoa procyanidins extracted from unfermented cocoa;

[0059]FIG. 3 shows several representative procyanidin structures;

[0060] FIGS. 4A-4E show representative HPLC chromatograms of fivefractions employed in screening for anti-cancer or antineoplasticactivity;

[0061]FIGS. 5 and 6A-6D show the dose-response relationship betweencocoa extracts and cancer cells ACHN (FIG. 5) and PC-3 (FIGS. 6A-6D)(fractional survival vs. dose, μg/mL); M&M2 F4/92, M&MA+E U12P1, M&MB+EY192P1, M&MC+E U12P2, M&MD+E U12P2;

[0062]FIGS. 7A to 7H show the typical dose response relationshipsbetween cocoa procyanidin fractions A, B, C, D, E, A+B, A+E, and A+D,and the PC-3 cell line (fractional survival vs. dose, μg/mL); MM-1A0212P3, MM-1 B 0162P1, MM-1 C 0122P3, MM-1 D 0122P3, MM-1 E 0292P8, MM-1A/B 0292P6, MM-1 A/E 0292P6, MM-1 A/D 0292P6;

[0063]FIGS. 8A to 8H show the typical dose response relationshipsbetween cocoa procyanidin fractions A, B, C, D, E, A+B, B+E, and D+E andthe KB Nasopharyngeal/HeLa cell line (fractional survival vs. dose,μg/mL); MM-1A092K3, MM-1 B 0212K5, MM-1 C 0162K3, MM-1 D 0212K5, MM-1 E0292K5, MM-1 A/B 0292K3, MM-1 B/E 0292K4, MM-1 D/E 0292K5;

[0064]FIGS. 9A to 9H show the typical dose response relationship betweencocoa procyanidin fractions A, B, C, D, E, B+D, A+E and D+E and theHCT-116 cell line (fractional survival vs. dose, μg/mL); MM-1 C 0192H5,D 0192H5, E 0192H5, MM-1 B&D 0262H2, A/E 0262H3, MM-1 D&E 0262H1;

[0065]FIGS. 10A to 10H show typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, B+D, C+D and A+E and the ACHNrenal cell line (fractional survival vs. dose, μg/mL); MM-1 A 092A5,MM-1 B 092A5, MM-1 C 0192A7, MM-1 D 0192A7, M&M1 E 0192A7, MM-1 B&D0302A6, MM-1 C&D 0302A6, MM-1 A&E 0262A6;

[0066]FIGS. 11A to 11H show typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, A+E, B+E and C+E and theA-549 lung cell line (fractional survival vs. dose, μg/mL); MM-1 A019258, MM-1 B 09256, MM-1 C 019259, MM-1 D 019258, MM-1 E 019258, A/E026254, MM-1 B&E 030255, MM-1 C&E N6255;

[0067]FIGS. 12A to 12H show typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, B+C, C+D and D+E and the SK-5melanoma cell line (fractional survival vs. dose μg/mL); MM-1 A 0212S4,MM-1 B 0212S4, MM-1 C 0212S4, MM-1 D 0212S4, MM-1 E N32S1, MM-1 B&CN32S2, MM-1 C&D N32S3, MM-1 D&E N32S3;

[0068]FIGS. 13A to 13H show typical dose response relationships betweencocoa procyanidin fractions A, B, C, D, E, B+C, C+E, and D+E and theMCF-7 breast cell line (fractional survival vs. dose, μg/mL); MM-1 AN22M4, MM-1 B N22M4, MM-1 C N22M4, MM-1 D N22M3, MM-1 E 0302M2, MM-1 B/C0302M4, MM-1 C&E N22M3, MM-1 D&E N22M3;

[0069]FIG. 14 shows typical dose response relationships for cocoaprocyanidin (particularly fraction D) and the CCRF-CEM T-cell leukemiacell line (cells/mL vs. days of growth; open circle is control, darkenedcircle is 125 μg fraction D, open inverted triangle is 250 μg fractionD, darkened inverted triangle is 500 μg fraction D);

[0070]FIG. 15A shows a comparison of the XTT and Crystal Violetcytotoxicity assays against MCF-7 p168 breast cancer cells treated withfraction D+E (open circle is XTT and darkened circle is Crystal Violet);

[0071]FIG. 15B shows a typical dose response curve obtained from MDAMB231 breast cell line treated with varying levels of crude polyphenolsobtained from UIT-1 cocoa genotype (absorbance (540 nm) vs. Days; opencircle is control, darkened circle is vehicle, open inverted triangle is250 μg/mL, darkened inverted triangle is 100 μg/mL, open square is 10μg/mL; absorbance of 2.0 is maximum of plate reader and may not benecessarily representative of cell number);

[0072]FIG. 15C shows a typical dose response curve obtained from PC-3prostate cancer cell line treated with varying levels of crudepolyphenols obtained from UIT-1 cocoa genotype (absorbance (540 nm) vs.Days; open circle is control, darkened circle is vehicle, open invertedtriangle is 250 μg/mL, darkened inverted triangle is 100 μg/mL and opensquare is 10 μg/mL);

[0073]FIG. 15D shows a typical dose-response curve obtained from MCF-7p168 breast cancer cell line treated with varying levels of crudepolyphenols obtained from UIT-1 cocoa genotype (absorbance (540 nm) vs.Days; open circle is control, darkened circle is vehicle, open invertedtriangle is 250 μg/mL, darkened inverted triangle is 100 μg/mL, opensquare is 10 μg/mL, darkened square is 1 μg/mL; absorbance of 2.0 ismaximum of plate reader and may not be necessarily representative ofcell number);

[0074]FIG. 15E shows a typical dose response curve obtained from Helacervical cancer cell line treated with varying levels of crudepolyphenols obtained from UIT-1 cocoa genotype (absorbance (540 nm) vs.Days; open circle is control, darkened circle is vehicle, open invertedtriangle is 250 μg/mL, darkened inverted triangle is 100 μg/mL, opensquare is 10 μg/mL; absorbance of 2.0 is maximum of plate reader and maynot be necessarily representative of cell number);

[0075]FIG. 15F shows cytotoxic effects against Hela cervical cancer cellline treated with different cocoa polyphenol fractions (absorbance (540nm) vs. Days; open circle is 100 μg/mL fractions A-E, darkened circle is100 μg/mL fractions A-C, open inverted triangle is 100 μg/mL fractionsD&E; absorbance of 2.0 is maximum of plate reader and not representativeof cell number);

[0076]FIG. 15G shows cytotoxic effects at 100 ul/mL against SKBR-3breast cancer cell line treated with different cocoa polyphenolfractions (absorbance (540 nm) vs. Days; open circle is fractions A-E,darkened circle is fractions A-C, open inverted triangle is fractionsD&E);

[0077]FIG. 15H shows typical dose-response relationships between cocoaprocyanidin fraction D+E on Hela cells (absorbance (540 nm) vs. Days;open circle is control, darkened circle is 100 μg/mL, open invertedtriangle is 75 μg/mL, darkened inverted triangle is 50 μg/mL, opensquare is 25 μg/mL, darkened square is 10 μg/mL; absorbance of 2.0 ismaximum of plate reader and is not representative of cell number);

[0078]FIG. 15I shows typical dose-response relationship between cocoaprocyanidin fraction D+E on SKBR-3 cells (absorbance (540 nm) vs. Days;open circle is control, darkened circle is 100 μg/mL, open invertedtriangle is 75 μg/mL, darkened inverted triangle is 50 μg/mL, opensquare is 25 μg/mL, darkened square is 10 μg/mL);

[0079]FIG. 15J shows typical dose-response relationships between cocoaprocyanidin fraction D+E on Hela cells using the Soft Agar Cloning assay(bar chart; number of colonies vs. control, 1, 10, 50, and 100 μg/mL);

[0080]FIG. 15K shows the growth inhibition of Hela cells when treatedwith crude polyphenol extracts obtained from eight different cocoagenotypes (% control vs. concentration, μg/mL; open circle is C-1,darkened circle is C-2, open inverted triangle is C-3, darkened invertedtriangle is C-4, open square is C-5, darkened square is C-6, opentriangle is C-7, darkened triangle is C-8; C-1=UF-12: hortirace=Trinitario and description is crude extracts of UF-12 (Brazil)cocoa polyphenols (decaffeinated/detheobrominated); C-2=NA-33: hortirace=Forastero and description is crude extracts of NA-33 (Brazil) cocoapolyphenols (decaffeinated/detheobrominated); C-3=EEG-48: hortirace=Forastero and description is crude extracts of EEG-48 (Brazil)cocoa polyphenols (decaffeinated/detheobrominated); C-4=unknown: hortirace=Forastero and description is crude extracts of unknown (W. African)cocoa polyphenols (decaffeinated/detheobrominated); C-5=UF-613: hortirace=Trinitario and description is crude extracts of UF-613 (Brazil)cocoa polyphenols (decaffeinated/detheobrominated); C-6=ICS-100: hortirace=Trinitario (to Nicaraguan Criollo ancestor) and description iscrude extracts of ICS-100 (Brazil) cocoa polyphenols(decaffeinated/detheobrominated); C-7=ICS-139: horti race=Trinitario(Nicaraguan Criollo ancestor) and description is crude extracts ofICS-139 (Brazil) cocoa polyphenols (decaffeinated/detheobrominated);C-8=UIT-1: horti race=Trinitario and description is crude extracts ofUIT-1 (Malaysia) cocoa polyphenols (decaffeinated/detheobrominated);

[0081]FIG. 15L shows the growth inhibition of Hela cells when treatedwith crude polyphenol extracts obtained from fermented cocoa beans anddried cocoa beans (stages throughout fermentation and sun drying; %control vs. concentration, μg/mL; open circle is day zero fraction,darkened circle is day 1 fraction, open inverted triangle is day 2fraction, darkened inverted triangle is day 3 fraction, open square isday 4 fraction and darkened square is day 9 fraction);

[0082]FIG. 15M shows the effect of enzymatically oxidized cocoaprocyanidins against Hela cells (dose response for polyphenol oxidasetreated crude cocoa polyphenol; % control vs. concentration, μg/mL;darkened square is crude UIT-1 (with caffeine and theobromine), opencircle crude UIT-1 (without caffeine and theobromine) and darkenedcircle is crude UIT-1 (polyphenol oxidase catalyzed);

[0083]FIG. 15N shows a representative semi-preparative reverse phaseHPLC separation for combined cocoa procyanidin fractions D and E;

[0084]FIG. 15O shows a representative normal phase semi-preparative HPLCseparation of a crude cocoa polyphenol extract;

[0085]FIG. 16 shows typical Rancimat Oxidation curves for cocoaprocyanidin extract and fractions in comparison to the syntheticantioxidants BHA and BHT (arbitrary units vs. time; dotted line andcross (+) is BHA and BHT; * is D-E; x is crude; open square is A-C; andopen diamond is control);

[0086]FIG. 17 shows a typical Agarose Gel indicating inhibition oftopoisomerase II catalyzed decatenation of kinetoplast DNA by cocoaprocyanidin fractions (Lane 1 contains 0.5 μg of marker (M)monomer-length kinetoplast DNA circles; Lanes 2 and 20 containkinetoplast DNA that was incubated with Topoisomerase II in the presenceof 4% DMSO, but in the absence of any cocoa procyanidins. (Control-C);Lanes 3 and 4 contain kinetoplast DNA that was incubated withTopoisomerase II in the presence of 0.5 and 5.0 μg/mL cocoa procyanidinfraction A; Lanes 5 and 6 contain kinetoplast DNA that was incubatedwith Topoisomerase II in the presence of 0.5 and 5.0 μg/mL cocoaprocyanidin fraction B; Lanes 7, 8, 9, 13, 14 and 15 are replicates ofkinetoplast DNA that was incubated with Topoisomerase II in the presenceof 0.05, 0.5 and 5.0 μg/mL cocoa procyanidin fraction D; Lanes 10, 11,12, 16, 17 and 18 are replicates of kinetoplast DNA that was incubatedwith Topoisomerase II in the presence of 0.05, 0.5, and 5.0 μg/mL cocoaprocyanidin fraction E; Lane 19 is a replicate of kinetoplast DNA thatwas incubated with Topoisomerase II in the presence of 5.0 μg/mL cocoaprocyanidin fraction E);

[0087]FIG. 18 shows dose response relationships of cocoa procyanidinfraction D against DNA repair competent and deficient cell lines(fractional survival vs. μg/mL; left side xrs-6 DNA Deficient RepairCell Line, MM-1 D D282X1; right side BR1 Competent DNA Repair Cell Line,MM-1 D D282B1);

[0088]FIG. 19 shows the dose-response curves for Adriamycin resistantMCF-7 cells in comparison to a MCF-7 p168 parental cell line whentreated with cocoa fraction D+E (% control vs. concentration, μg/mL;open circle is MCF-7 p168; darkened circle is MCF-7 ADR);

[0089]FIGS. 20A and B show the dose-response effects on Hela and SKBR-3cells when treated at 100 μg/mL and 25 μg/mL levels of twelve fractionsprepared by Normal phase semi-preparative HPLC (bar chart, % control vs.control and fractions 1-12);

[0090]FIG. 21 shows a normal phase HPLC separation of crude, enrichedand purified pentamers from cocoa extract;

[0091]FIGS. 22A, B and C show MALDI-TOF/MS of pentamer enrichedprocyanidins, and of Fractions A-C and of Fractions D-E, respectively;

[0092]FIG. 23A shows an elution profile of oligomeric procyanidinspurified by modified semi-preparative HPLC;

[0093]FIG. 23B shows an elution profile of a trimer procyanidin bymodified semi-preparative HPLC;

[0094] FIGS. 24A-D each show energy minimized structures of all (4-8)linked pentamers based on the structure of epicatechin;

[0095]FIG. 25A shows relative fluorescence of epicatechin upon thiolysiswith benzylmercapten;

[0096]FIG. 25B shows relative fluorescence of catechin upon thiolysiswith benzylmercapten;

[0097]FIG. 25C shows relative fluorescence of dimers (B2 and B5) uponthiolysis with benzylmercapten;

[0098]FIG. 26A shows relative fluorescence of dimer upon thiolysis;

[0099]FIG. 26B shows relative fluorescence of B5 dimer upon thiolysis ofdimer and subsequent desulphurization;

[0100]FIG. 27A shows the relative tumor volume during treatment of MDAMB 231 nude mouse model treated with pentamer;

[0101]FIG. 27B shows the relative survival curve of pentamer treated MDA231 nude mouse model;

[0102]FIG. 28 shows the elution profile from halogen-free analyticalseparation of acetone extract of procyanidins from cocoa extract;

[0103]FIG. 29 shows the effect of pore size of stationary phase fornormal phase HPLC separation of procyanidins;

[0104]FIG. 30A shows the substrate utilization during fermentation ofcocoa beans;

[0105]FIG. 30B shows the metabolite production during fermentation;

[0106]FIG. 30C shows the plate counts during fermentation of cocoabeans;

[0107]FIG. 30D shows the relative concentrations of each component infermented solutions of cocoa beans;

[0108]FIG. 31 shows the acetylcholine-induced relaxation of NO-relatedphenylephrine-precontracted rat aorta;

[0109]FIG. 32 shows the blood glucose tolerance profiles from varioustest mixtures;

[0110] FIGS. 33A-B show the effects of indomethacin on COX-1 and COX-2activities;

[0111] FIGS. 34A-B show the correlation between the degree ofpolymerization and IC₅₀ vs. COX-1/COX-2 (μM);

[0112]FIG. 35 shows the correlation between the effects of compounds onCOX-1 and COX-2 activities expressed as μM;

[0113] FIGS. 36A-V show the IC₅₀ values (μM) of samples containingprocyanidins with COX-1/COX-2;

[0114]FIG. 37 shows the purification scheme for the isolation ofprocyanidins from cocoa;

[0115]FIGS. 38A to 38P shows the preferred structures of the pentamer;

[0116] FIGS. 39A-AA show a library of stereoisomers of pentamers;

[0117] FIGS. 40A-B show 70 minute gradients for normal phase HPLCseparation of procyanidins, detected by UV and fluorescence,respectively;

[0118] FIGS. 41A-B show 30 minute gradients for normal phase HPLCseparation of procyanidins, detected by UV and fluorescence,respectively;

[0119]FIG. 42 shows a preparation normal phase HPLC separation ofprocyanidins;

[0120] FIGS. 43A-G show CD (circular dichroism) spectra of procyanidindimers, trimers, tetramers, pentamers, hexamers, heptamers and octamers,respectively;

[0121]FIG. 44A shows the structure and ¹H/¹³C NMR data for epicatechin;

[0122] FIGS. 44B-F show the APT, COSY, XHCORR, ¹H and ¹³C NMR spectrafor epicatechin;

[0123]FIG. 45A shows the structure and ¹H/¹³C NMR data for catechin;

[0124] FIGS. 45B-E show the ¹H, APT, XHCORR and COSY NMR spectra forcatechin;

[0125]FIG. 46A shows the structure and ¹H/¹³C NMR data for B2 dimer;

[0126] FIGS. 46B-G show the ¹³C, APT, ¹H, HMQC, COSY and HOHAHA NMRspectra for the B2 dimer;

[0127]FIG. 47A shows the structure and ¹H/¹³C NMR data for B5 dimer;

[0128] FIGS. 47B-G show the ¹H, ¹³C, APT, COSY, HMQC and HOHAHA NMRspectra for B5 dimer;

[0129] FIGS. 48A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra forepicatechin/catechin trimer;

[0130] FIGS. 49A-D show the ¹H, COSY, HMQC and HOHAHA NMR spectra forepicatechin trimer;

[0131]FIGS. 50A and B show the effects of cocoa procyanidin fraction Aand C, respectively, on blood pressure; blood pressure levels decreasedby 21.43% within 1 minute after administration of fraction A, andreturned to normal after 15 minutes, while blood pressure decreased by50.5% within 1 minute after administration of fraction C, and returnedto normal after 5 minutes;

[0132]FIG. 51 shows the effect of cocoa procyanidin fractions onarterial blood pressure in anesthetized guinea pigs;

[0133]FIG. 53 shows the effect of bradykinin on NO production by HUVEC;

[0134]FIG. 54 shows the effect of cocoa procyanidin fractions onmacrophage NO production by HUVEC;

[0135]FIG. 55 shows the effect of cocoa procyanidin fractions onnacrophage NO production;

[0136]FIG. 56 shows the effect of cocoa procyanidin fraction on LPSinduced and gamma-Interferon primed macrophages.

[0137]FIG. 57 shows a micellar electrokinetic capillary chromatographicseparation of cocoa procyanidin oligomers;

[0138] FIGS. 58 A-F show MALDI-TOF mass spectra for Cu⁺²-, Zn⁺²-, Fe⁺²-,Fe⁺³-, Ca⁺²-, and Mg⁺²-ions, respectively, completed to a trimer;

[0139]FIG. 59 shows a MALDI-TOF mass spectrum of cocoa procyanidinoligomers (tetramers to octadecamers);

[0140]FIG. 60 shows the dose-response relationship of cocoa procyanidinoligomers and the feline FeA lymphoblastoid cell line producing leukemiavirus;

[0141]FIG. 61 shows the dose-response relationship of cocoa procyanidinoligomers and the feline CRFK normal kidney cell line;

[0142]FIG. 62 shows the dose-response relationship of cocoa procyanidinoligomers and the canine MDCK normal kidney line;

[0143]FIG. 63 shows the dose-response relationship between cocoaprocyanidin oligomers and the canine GH normal kidney cell line;

[0144]FIG. 64 shows time-temperature effects on hexamer hydrolysis; and

[0145]FIG. 65 shows time-temperature effects on trimer formation.

DETAILED DESCRIPTION Compounds of the Invention

[0146] As discussed above, it has now been surprisingly found that cocoaextracts or compounds derived therefrom exhibit anti-cancer, anti-tumoror antineoplastic activity, antioxidant activity, inhibit DNAtopoisomerase II enzyme and oxidative damage to DNA, and haveantimicrobial, cyclo-oxygenase and/or lipoxygenase, NO or NO-synthase,apoptosis, platelet aggregation and blood or in vivo glucose, modulatingactivities, as well as efficacy as a non-steroidal antiinflammatoryagent.

[0147] The extracts, compounds or combination of compounds derivedtherefrom are generally prepared by reducing cocoa beans to a powder,defatting the powder, and extracting and purifying the activecompound(s) from the defatted powder. The powder can be prepared byfreeze-drying the cocoa beans and pulp, depulping and dehulling thefreeze-dried cocoa beans and grinding the dehulled beans. The extractionof active compound(s) can be by solvent extraction techniques. Theextracts comprising the active compounds can be purified, e.g., to besubstantially pure, for instance, by gel permeation chromatography or bypreparative High Performance Liquid Chromatography (HPLC) techniques orby a combination of such techniques.

[0148] With reference to the isolation and purification of the compoundsof the invention derived from cocoa, it will be understood that anyspecies of Theobroma, Herrania or inter- and intra-species crossesthereof may be employed. In this regard, reference is made to Schultes,“Synopsis of Herrania,” Journal of the Arnold Arboretum, Vol. XXXIX, pp.217 to 278, plus plates I to XVII (1985), Cuatrecasas, “Cocoa and ItsAllies, A Taxonomic Revision of the Genus Theobroma,” Bulletin of theUnited States National Museum, Vol. 35, part 6, pp. 379 to 613, plusplates 1 to 11 (Smithsonian Institution, 1964), and Addison, et al.,“Observations on the Species of the Genus Theobroma Which Occurs in theAmazon,” Bol. Tech. Inst. Agronomico de Nortes, 25(3) (1951).

[0149] Additionally, Example 25 lists the heretofore never reportedconcentrations of the inventive compounds found in Theobroma andHerrania species and their inter- and intra-species crosses; and Example25 also describes methods of modulating the amounts of the inventivecompounds which may be obtained from cocoa by manipulating cocoafermentation conditions.

[0150] An outline of the purification protocol utilized in the isolationof substantially pure procyanidins is shown in FIG. 37. Steps 1 and 2 ofthe purification scheme are described in Examples 1 and 2; steps 3 and 4are described in Examples 3, 13 and 23; step 5 is described in Examples4 and 14; and step 6 is described in Examples 4, 14 and 16. The skilledartisan would appreciate and envision modifications in the purificationscheme outlined in FIG. 37 to obtain the active compounds withoutdeparting from the spirit or scope thereof and without undueexperimentation.

[0151] The extracts, compounds and combinations of compounds derivedtherefrom having activity, without wishing to necessarily be bound byany particular theory, have been identified as cocoa polyphenol(s), suchas procyanidins. These cocoa procyanidins have significant anti-cancer,anti-tumor or antineoplastic activity; antioxidant activity; inhibit DNAtopoisomerase II enzyme and oxidative damage to DNA; possessantimicrobial activity; have the ability to modulate cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregationand blood or in vivo glucose, and have efficacy as non-steroidalantiinflammatory agents.

[0152] The present invention provides a compound of the formula:

[0153] wherein:

[0154] n is an integer from 2 to 18, e.g., 3 to 12, such that there is afirst monomeric unit A, and a plurality of other monomeric units;

[0155] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0156] position 4 is alpha or beta stereochemistry;

[0157] X, Y and Z represent positions for bonding between monomericunits, with the provisos that as to the first monomeric unit, bonding ofanother monomeric unit thereto is at position 4 and Y=Z=hydrogen, and,that when not for bonding monomeric units, X, Y and Z are hydrogen, orZ, Y are sugar and X is hydrogen, or X is alpha or beta sugar and Z, Yare hydrogen, or combinations thereof. The compound can have n as 5 to12, and certain preferred compounds have n as 5. The sugar can beselected from the group consisting of glucose, galactose, xylose,rhamnose, and arabinose. The sugar of any or all of R, X, Y and Z canoptionally be substituted with a phenolic moiety via an ester bond.

[0158] Thus, the invention can provide a compound of the formula:

[0159] wherein:

[0160] n is an integer from 2 to 18, e.g., 3 to 12, advantageously 5 to12, and preferably n is 5, such that there is a first monomeric unit A,

[0161] and a plurality of other monomeric units of A;

[0162] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0163] position 4 is alpha or beta stereochemistry;

[0164] X, Y and Z represent positions for bonding between monomericunits, with the provisos that as to the first monomeric unit, bonding ofanother monomeric unit thereto is at position 4 and Y=Z=hydrogen, and,that when not for bonding monomeric units, X, Y and Z are hydrogen or Z,Y are sugar and X is hydrogen, or X is alpha or beta sugar and Z and Yare hydrogen, or combinations thereof; and

[0165] said sugar is optionally substituted with a phenolic moiety viaan ester bond.

[0166] Accordingly, the present invention provides a polymeric compoundof the formula A_(n), wherein A is a monomer having the formula:

[0167] wherein

[0168] n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and at least one or a plurality of additionalmonomeric units;

[0169] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0170] bonding between adjacent monomers takes place at positions 4, 6or 8;

[0171] a bond of an additional monomeric unit in position 4 has α or βstereochemistry;

[0172] X, Y and Z are selected from the group consisting of monomericunit A, hydrogen, and a sugar, with the provisos that as to the at leastone terminal monomeric unit, bonding of the additional monomeric unitthereto (i.e., the bonding of the monomeric unit adjacent the terminalmonomeric unit) is at position 4 and optionally, Y=Z=hydrogen;

[0173] the sugar is optionally substituted with a phenolic moiety at anyposition, for instance via an ester bond, and pharmaceuticallyacceptable salts or derivatives thereof (including oxidation products).

[0174] In preferred embodiments, n can be 3 to 18, 2 to 18, 3 to 12,e.g., 5 to 12; and, advantageously, n is 5. The sugar is selected fromthe group consisting of glucose, galactose, xylose, rhamnose andarabinose. The sugar of any or all of R, X, Y and Z can optionally besubstituted at any position with a phenolic moiety via an ester bond.The phenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.

[0175] Additionally, the present invention provides a polymeric compoundof the formula A_(n), wherein A is a monomer having the formula:

[0176] wherein

[0177] n is an integer from 2 to 18, e.g., 3 to 18, advantageously 3 to12, e.g., 5 to 12, preferably n is 5;

[0178] R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar;

[0179] adjacent monomers bind at position 4 by (4→6) or (4→8) ;

[0180] each of X, Y and Z is H, a sugar or an adjacent monomer, with theprovisos that if X and Y are adjacent monomers, Z is H or sugar and if Xand Z are adjacent monomers, Y is H or sugar, and that as to at leastone of the two terminal monomers, bonding of the adjacent monomer is atposition 4 and optionally, Y=Z=hydrogen;

[0181] a bond at position 4 has α or β stereochemistry;

[0182] the sugar is optionally substituted with a phenolic moiety at anyposition, for instance, via an ester bond,

[0183] and pharmaceutically acceptable salts or derivatives thereof(including oxidation products).

[0184] With regard to the recitation of “at least one terminal monomericunit A”, it will be understood that the inventive compounds have twoterminal monomeric units, and that the two terminal monomeric unit A maybe the same or different. Additionally, it will be understood that therecitation of “at least one terminal monomeric unit A” includesembodiments wherein the terminal monomeric unit A is referred to as a“first monomeric unit”, with the recitation of “first monomeric unit”relating to that monomer to which other monomeric units are added,resulting in a polymeric compound of the formula A_(n). Moreover, withregard to the at least one of the two terminal monomers, bonding of theadjacent monomer is at position 4 and optionally, Y=Z=hydrogen.

[0185] As to the recitation of the term “combinations thereof”, it willbe understood that one or more of the inventive compounds may be usedsimultaneously, e.g., administered to a subject in need of treatment ina formulation comprising one or more inventive compounds.

[0186] The inventive compounds or combinations thereof display theutilities noted above for cocoa extracts; and throughout the disclosure,the term “cocoa extract” may be substituted by compounds of theinvention or combinations thereof, such that it will be understood thatthe inventive compounds or combinations thereof can be cocoa extracts.

[0187] The term “oligomer”, as used herein, refers to any compounds orcombinations thereof of the formula presented above, wherein n is 2through 18. When n is 2, the oligomer is termed a “dimer”; when n is 3,the oligomer is termed a “trimer”; when n is 4, the oligomer is termed a“tetramer”; when n is 5, the oligomer is termed a “pentamer”; andsimilar recitations may be designated for oligomers having n up to andincluding 18, such that when n is 18, the oligomer is termed an“octadecamer”.

[0188] The inventive compounds or combinations thereof can be isolated,e.g., from a natural source such as any species of Theobroma, Herraniaor inter- or intra-species crosses thereof; or, the inventive compoundsor combinations thereof can be purified, e.g., compounds or combinationsthereof can be substantially pure; for instance, purified to apparenthomogeneity. Purity is a relative concept, and the numerous Examplesdemonstrate isolation of inventive compounds or combinations thereof, aswell as purification thereof, such that by methods exemplified a skilledartisan can obtain a substantially pure inventive compound orcombination thereof, or purify them to apparent homogeneity (e.g.,purity by separate, distinct chromatographic peak). Considering theExamples (e.g., Example 37), a substantially pure compound orcombination of compounds is at least about 40% pure, e.g., at leastabout 50% pure, advantageously at least about 60% pure, e.g., at leastabout 70% pure, more advantageously at least about 75-80% pure,preferably, at least about 90% pure, more preferably greater than 90%pure, e.g., at least 90-95% pure, or even purer, such as greater than95% pure, e.g., 95-98% pure.

[0189] Further, examples of the monomeric units comprising the oligomersused herein are (+)-catechin and (−)-epicatechin, abbreviated C and EC,respectively. The linkages between adjacent monomers are from position 4to position 6 or position 4 to position 8; and this linkage betweenposition 4 of a monomer and position 6 and 8 of the adjacent monomericunits is designated herein as (4→6) or (4→8). There are four possiblestereochemical linkages between position 4 of a monomer and position 6and 8 of the adjacent monomer; and the stereochemical linkages betweenmonomeric units is designated herein as (4α→6) or (4α→6) or (4α→8) or(4β→8). When C is linked to another C or EC, the linkages are designatedherein as (4α→6) or (4α→8). When EC is linked to another C or EC, thelinkages are designated herein as (4β→6) or (4β→8).

[0190] Examples of compounds eliciting the activities cited aboveinclude dimers, EC-(4β→8)-EC and EC-(4β→6)-EC, wherein EC-(4β→8)-EC ispreferred; trimers [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC,wherein [EC-(4β→8)]₂-EC is preferred; tetramers [EC-(4β→8)]₃-EC,[EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, wherein [EC-(4β→8)]₃-EC ispreferred; and pentamers [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)1₃-EC-(4β→6)-C, wherein the3-position of the pentamer terminal monomeric unit is optionallyderivatized with a gallate or β-D-glucose; [EC-(4β→8)]₄-EC is preferred.

[0191] Additionally, compounds which elicit the activities cited abovealso include hexamers to dodecamers, examples of which are listed below:

[0192] A hexamer, wherein one monomer (C or EC) having linkages toanother monomer (4β→8) or (4β→6) for EC linked to another EC or C, and(4α→8) or (4α→6) for C linked to another C or EC; followed by a (4β→8)linkage to a pentamer compound listed above, e.g., [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, wherein the 3-position of the hexamer terminalmonomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the hexamer is [EC-(4β→8)]₅-EC;

[0193] A heptamer, wherein any combination of two monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,(EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, wherein the 3-position of the heptamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the heptamer is [EC-(4β→8)]₆-EC;

[0194] An octamer, wherein any combination of three monomers (C and/orEC) having linkages to one another (4β→8) or (4β→6) for EC linked toanother EC or C, and (4α→8) or (4α→6) for C linked to another C or EC;followed by a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, wherein the 3-position of the octamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the octamer is [EC-(4β→8)]₇-EC;

[0195] A nonamer, wherein any combination of four monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, wherein the 3-position of the nonamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the nonamer is [EC-(4β→8)]₈-EC;

[0196] A decamer, wherein any combination of five monomers (C and/or EC)having linkages to one another (4β→8) or (4β→6) for EC linked to anotherEC or C, and (4α→8) or (4α→6) for C linked to another C or EC; followedby a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,and [EC-(4β→8)]₈-EC-(4β→6)-C, wherein the 3-position of the decamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the decamer is [EC-(4β→8)]₉-EC;

[0197] An undecamer, wherein any combination of six monomers (C and/orEC) having linkages to one another (4β→8) or (4β→6) for EC linked toanother EC or C, and (4α→8) or (4α→6) for C linked to another C or EC;followed by a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,and [EC-(4β→8)]₉-EC-(4β→6)-C, wherein the 3-position of the undecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the undecamer is[EC-(4β→8)]₁₀-EC; and

[0198] A dodecamer, wherein any combination of seven monomers (C and/orEC) having linkages to one another (4β→8) or (4β→6) for EC linked toanother EC or C, and (4α→8) or (4α→6) for C linked to another C or EC;followed by a (4β→8) linkage to a pentamer compound listed above, e.g.,[EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C,and [EC-(4β→8)]₁₀-EC-(4β→6)-C, wherein the 3-position of the dodecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose; in a preferred embodiment, the dodecamer is[EC-(4β→8)]₁₁-EC.

[0199] It will be understood from the detailed description that theaforementioned list is exemplary and provided as an illustrative sourceof several non-limiting examples of compounds of the invention, which isby no means an exhaustive list of the inventive compounds encompassed bythe present invention.

[0200] Examples 3A, 3B, 4, 14, 23, 24, 30 and 34 describe methods toseparate the compounds of the invention. Examples 13, 14A-D and 16describe methods to purify the compounds of the invention. Examples 5,15, 18, 19, 20 and 29 describe methods to identify compounds of theinvention. FIGS. 38A-P and 39A-AA illustrate a stereochemical libraryfor representative pentamers of the invention. Example 17 describes amethod to molecularly model the compounds of the invention. Example 36provides evidence for higher oligomers in cocoa, wherein n is 13 to 18.

[0201] Furthermore, while the invention is described with respect tococoa extracts preferably comprising cocoa procyanidins, from thisdisclosure the skilled organic chemist will appreciate and envisionsynthetic routes to obtain and/or prepare the active compounds (seee.g., Example 11). Accordingly, the invention comprehends syntheticcocoa polyphenols or procyanidins or their derivatives and/or theirsynthetic precursors which include, but are not limited to glycosides,gallates, esters, etc. and the like. That is, the inventive compoundscan be prepared from isolation from cocoa or from any species within theTheobroma or Herrania genera, as well as from synthetic routes; andderivatives and synthetic precursors of the inventive compounds such asglycosides, gallates, esters, etc. are included in the inventivecompounds. Derivatives can also include compounds of the above formulaewherein a sugar or gallate moiety is on the terminal monomer atpositions Y or Z, or a substituted sugar or gallate moiety is on theterminal monomer at Y or Z.

[0202] For example, Example 8, Method C describes the use of cocoaenzymes to oxidatively modify the compounds of the invention orcombinations thereof to elicit improved cytotoxicity (see FIG. 15M)against certain cancer cell lines. The invention includes the ability toenzymatically modify (e.g., cleavage or addition of a chemicallysignificant moiety) the compounds of the invention, e.g., enzymaticallywith polyphenol oxidase, peroxidase, catalase combinations, and/orenzymes such as hydrolases, esterases, reductases, transferases, and thelike and in any combination, taking into account kinetic andthermodynamic factors (see also Example 41 regarding hydrolysis).

[0203] With regard to the synthesis of the inventive compounds, theskilled artisan will be able to envision additional routes of synthesis,based on this disclosure and the knowledge in the art, without undueexperimentation. For example, based upon a careful retrosyntheticanalysis of the polymeric compounds, as well as the monomers. Forinstance, given the phenolic character of the inventive compounds, theskilled artisan can utilize various methods of selectiveprotection/deprotection, coupled with organometallic additions, phenoliccouplings and photochemical reactions, e.g., in a convergent, linear orbiomimetic approach, or combinations thereof, together with standardreactions known to those well-versed in the art of synthetic organicchemistry, as additional synthetic methods for preparing the inventivecompounds, without undue experimentation. In this regard, reference ismade to W. Carruthers, Some Modern Methods of Organic Synthesis, 3rded., Cambridge University Press, 1986, and J. March, Advanced OrganicChemistry, 3rd ed., John Wiley & Sons, 1985, van Rensburg et al., Chem.Comm., 24: 2705-2706 (Dec. 21, 1996), Ballenegger et al., (Zyma SA)European Patent 0096 007 B1, and documents in the References sectionbelow, all of which are hereby incorporated herein by reference.

Utilities of Compounds of the Invention

[0204] With regard to the inventive compounds, it has been surprisinglyfound that the inventive compounds have discrete activities, and assuch, the inventive compounds have broad applicability to the treatmentof a variety of disease conditions, discussed hereinbelow.

COX/LOX-Associated Utilities

[0205] Atherosclerosis, the most prevalent of cardiovascular diseases,is the principle cause of heart attack, stroke and vascular circulationproblems. Atherosclerosis is a complex disease which involves many celltypes, biochemical events and molecular factors. There are severalaspects of this disease, its disease states and disease progressionwhich are distinguished by the interdependent consequences of LowDensity Lipoprotein (LDL) oxidation, cyclo-oxygenase (COX)/lipoxygenase(LOX) biochemistry and Nitric Oxide (NO) biochemistry.

[0206] Clinical studies have firmly established that the elevated plasmaconcentrations of LDL are associated with accelerated atherogenesis. Thecholesterol that accumulates in atherosclerotic lesions originateprimarily in plasma lipoproteins, including LDL. The oxidation of LDL isa critical event in the initiation of atheroma formation and isassociated with the enhanced production of superoxide anion radical(O₂•—). Oxidation of LDL by O₂•— or other reactive species (e.g., •OH,ONOO•—, lipid peroxy radical, copper ion, and iron based proteins)reduces the affinity of LDL for uptake in cells via receptor mediatedendocytosis. Oxidatively modified LDLs are then rapidly taken up bymacrophages which subsequently transform into cells closely resemblingthe “foam cells” observed in early atherosclerotic lesions.

[0207] Oxidized lipoproteins can also promote vascular injury throughthe formation of lipid hydroperoxides within the LDL particle. Thisevent initiates radical chain oxidation reactions of unsaturated LDLlipids, thus producing more oxidized LDL for macrophage incorporation.

[0208] The collective accumulation of foam cells engorged with oxidizedLDL from these processes results in early “fatty streak” lesions, whicheventually progress to the more advanced complex lesions ofatherosclerosis leading to coronary disease.

[0209] As discussed generally by Jean Marx at page 320 of Science, Vol.265 (Jul. 15, 1994), each year about 330,000 patients in the UnitedStates undergo coronary and/or peripheral angioplasty, a proceduredesigned to open up blood vessels, e.g., coronary arteries, clogged bydangerous atherosclerotic plaques (atherosclerosis) and thereby restorenormal blood flow. For a majority of these patients, the operation worksas intended. Nearly 33% of these patients (and maybe more by someaccounts), however, develop restenosis, wherein the treated arteriesbecome quickly clogged again. These patients are no better off, andsometimes worse off, than they were before angioplasty. Excessiveproliferation of smooth muscle cells (SMCs) in blood vessel wallscontributes to restenosis. Increased accumulation of oxidized LDL withinlesion SMCs might contribute to an atherogenic-related process likerestenosis. Zhou et al., “Association Between Prior CytomegalovirusInfection And The Risk Of Restenosis After Coronary Atherectomy,” Aug.29, 1996, New England Journal of Medicine, 335:624-630, and documentscited therein, all incorporated herein by reference. Accordingly,utility of the present invention with respect to atherosclerosis canapply to restenosis.

[0210] With regard to the inhibition by the inventive compounds ofcyclooxygenases (COX; prostaglandin endoperoxide synthase), it is knownthat cyclooxygenases are central enzymes in the production ofprostaglandins and other arachidonic acid metabolites (i.e.,eicosanoids) involved in many physiological processes. COX-1 is aconstitutive enzyme expressed in many tissues, including platelets,whereas COX-2, a second isoform of the enzyme, is inducible by variouscytokines, hormones and tumor promoters. COX 1 produces thromboxane A2,which is involved in platelet aggregation, which in turn is involved inthe progression of atherosclerosis. Its inhibition is the basis for theprophylactic effects on cardiovascular disease.

[0211] The activity of COX-1 and COX-2 is inhibited by aspirin and othernonsteroidal antiinflammatory drugs (NSAIDs), and the gastric sideeffects of NSAIDs are believed to be associated with the inhibition ofCOX-1. Moreover, it has been found that patients taking NSAIDs on aregular basis have a 40 to 50% lower risk of contracting colorectalcancer when compared to persons not being administered these type ofmedications; and COX-2 mRNA levels are markedly increased in 86% ofhuman colorectal adenocarcinomas.

[0212] One significant property of COX-2 expressing cell lines is theenhanced expression of genes which participate in the modulation ofapoptosis, i.e., programmed cell death. Several NSAIDs have beenimplicated in increased cell death and the induction of apoptosis inchicken embryo fibroblasts.

[0213] Cellular lipoxygenases are also involved in the oxidativemodification of LDL through the peroxidation of unsaturated lipids. Thegeneration of lipid peroxy radicals contributes to the further radicalchain oxidation of unsaturated LDL lipids, producing more oxidized LDLfor macrophage incorporation.

[0214] It has been surprisingly found that the inventive compounds haveutility in the treatment of diseases associated with COX/LOX. In Example28, COX was inhibited by individual inventive compounds atconcentrations similar to a known NSAID, indomethacin.

[0215] For COX inhibition, the inventive compounds are oligomers, wheren is 2 to 18. In a preferred embodiment, the inventive compounds areoligomers where n is 2 to 10, and more preferably, the inventivecompounds are oligomers where n is 2 to 5.

[0216] Examples of compounds eliciting the inhibitory activity withrespect to COX/LOX cited above include dimers, trimers, tetramers andpentamers, discussed above.

[0217] Hence, given the significant inhibitory potency of the inventivecompounds on COX-2, coupled with the cytotoxic effects on a putativeCOX-2 expression colon cancer cell line, the inventive compounds possessapoptotic activity as inhibitors of the multistep progression leading tocarcinomas, as well as activity as members of the NSAID family ofmedications possessing a broad spectrum of prophylactic activities (see,e.g., Example 8, FIGS. 9D to 9H).

[0218] Further, prostaglandins, the penultimate products of the COXcatalyzed conversion of arachidonic acid to prostaglandin H₂, areinvolved in inflammation, pain, fever, fetal development, labor andplatelet aggregation. Therefore, the inventive compounds are efficaciousfor the same conditions as NSAIDs, e.g., against cardiovascular disease,and stroke, etc. (indeed, the inhibition of platelet COX-1, whichreduces thromboxane A₂ production, is the basis for the prophylacticeffects of aspirin on cardiovascular disease).

[0219] Inflammation is the response of living tissues to injury. Itinvolves a complex series of enzyme activation, mediator release,extravasation of fluid, cell migration, tissue breakdown and repair.Inflammation is activated by phospholipase A₂, which liberatesarachidonic acid, the substrate for COX and LOX enzymes. COX convertsarachidonic acid to the prostaglandin PGE₂, the major eicosanoiddetected in inflammatory conditions ranging from acute edema to chronicarthritis. Its inhibition by NSAIDs is a mainstay for treatment.

[0220] Arthritis is one of the rheumatic diseases which encompass a widerange of diseases and pathological processes, most of which affect jointtissue. The basic structure affected by these diseases is the connectivetissue which includes synovial membranes, cartilage, bone, tendons,ligaments, and interstitial tissues. Temporary connective tissuesyndromes include sprains and strains, tendonitis, and tendon sheathabnormalities. The most serious forms of arthritis are rheumatoidarthritis, osteoarthritis, gout and systemic lupus erythematosus.

[0221] In addition to the rheumatic diseases, other diseases arecharacterized by inflammation. Gingivitis and periodontitis follows apathological picture resembling rheumatoid arthritis. Inflammatory boweldisease refers to idiopathic chronic inflammatory conditions of theintestine, ulcerative colitis and Crohn's disease. Spondylitis refers tochronic inflammation of the joints of the spine. There is also a highincidence of osteoarthritis associated with obesity.

[0222] Thus, the inventive compounds have utility in the treatment ofconditions involving inflammation, pain, fever, fetal development, laborand platelet aggregation.

[0223] The inhibition of COX by the inventive compounds would alsoinhibit the formation of postaglandins, e.g., PGD₂, PGE₂. Thus, theinventive compounds have utility in the treatment of conditionsassociated with prostaglandin PGD₂, PGE₂.

NO-Associated Utilities

[0224] Nitric oxide (NO) is known to inhibit platelet aggregation,monocyte adhesion and chemotaxis, and proliferation of vascular smoothmuscle tissue which are critically involved in the process ofatherogenesis. Evidence supports the view that NO is reduced inatherosclerotic tissues due to its reaction with oxygen free radicals.The loss of NO due to these reactions leads to increased platelet andinflammatory cell adhesion to vessel walls to further impair NOmechanisms of relaxation. In this manner, the loss of NO promotesatherogenic processes, leading to progressive disease states.

[0225] Hypertension is a leading cause of cardiovascular diseases,including stroke, heart attack, heart failure, irregular heart beat andkidney failure. Hypertension is a condition where the pressure of bloodwithin the blood vessels is higher than normal as it circulates throughthe body. When the systolic pressure exceeds 150 mm Hg or the diastolicpressure exceeds 90 mm Hg for a sustained period of time, damage is doneto the body. For example, excessive systolic pressure can rupture bloodvessels anywhere. When it occurs within the brain, a stroke results. Itcan also cause thickening and narrowing of the blood vessels which canlead to atherosclerosis. Elevated blood pressure can also force theheart muscle to enlarge as it works harder to overcome the elevatedresting (diastolic) pressure when blood is expelled. This enlargementcan eventually produce irregular heart beats or heart failure.Hypertension is called the “silent killer” because it causes no symptomsand can only be detected when blood pressure is checked.

[0226] The regulation of blood pressure is a complex event where onemechanism involves the expression of constitutive Ca⁺²/calmodulindependent form of nitric oxide synthase (NOS), abbreviated eNOS. NOproduced by this enzyme produces muscle relaxation in the vessel(dilation), which lowers the blood pressure. When the normal level of NOproduced by eNOS is not produced, either because production is blockedby an inhibitor or in pathological states, such as atherosclerosis, thevascular muscles do not relax to the appropriate degree. The resultingvasoconstriction increases blood pressure and may be responsible forsome forms of hypertension.

[0227] Vascular endothelial cells contain eNOS. NO synthesized by eNOSdiffuses in diverse directions, and when it reaches the underlyingvascular smooth muscle, NO binds to the heme group of guanylyl cyclase,causing an increase in cGMP. Increased cGMP causes a decrease inintracellular free Ca⁺². Cyclic GMP may activate a protein kinase thatphosphorylates Ca⁺² transporters, causing Ca⁺² to be sequestered inintracellular structures in the muscle cells. Since muscle contractionrequires Ca⁺², the force of the contraction is reduced as the Ca⁺²concentration declines. Muscle relaxation allows the vessel to dilate,which lowers the blood pressure. Inhibition of eNOS therefore causesblood pressure to increase.

[0228] When the normal level of NO is not produced, either becauseproduction is blocked by administration of an NOS inhibitor or possibly,in pathological states, such as atherosclerosis, the vascular muscles donot relax to the appropriate degree. The resulting vasoconstrictionincreases blood pressure and may be responsible for some forms ofhypertension. There is considerable interest in finding therapeutic waysto increase the activity of eNOS in hypertensive patients, but practicaltherapies have not been reported. Pharmacological agents capable ofreleasing NO, such as nitroglycerin or isosorbide dinitrate, remainmainstays of vasorelaxant therapy.

[0229] Although the inventive compounds inhibit the oxidation of LDL,the more comprehensive effects of these compounds is theirmultidimensional effects on atherosclerosis via NO. NO modulation by theinventive compounds brings about a collage of beneficial effects,including the modulation of hypertension, lowering NO affectedhypercholesterolemia, inhibiting platelet aggregation and monocyteadhesion, all of which are involved with the progression ofatherosclerosis.

[0230] The role of NO in the immune system is different from itsfunction in blood vessels. Macrophages contain a form of NOS that isinducible, rather than constitutive, referred to as iNOS. Transcriptionof the iNOS gene is controlled both positively and negatively by anumber of biological response modifiers called cytokines. The mostimportant inducers are gamma-interferon, tumor necrosis factor,interleukin-1, interleukin-2 and lipopolysaccharide (LPS), which is acomponent of the cell walls of gram negative bacteria. Stimulatedmacrophages produce enough NO to inhibit ribonuclease reductase, theenzyme that converts ribonucleotides to the deoxyribonucleotidesnecessary for DNA synthesis. Inhibition of DNA synthesis may be animportant way in which macrophages and other tissues possessing iNOS caninhibit the growth of rapidly dividing tumor cells or infectiousbacteria.

[0231] With regard to the effects of NO and infectious bacteria,microorganisms play a significant role in infectious processes whichreflect body contact and injury, habits, profession, environment of theindividual, as well as food borne diseases brought about by improperstorage, handling and contamination.

[0232] The inventive compounds, combinations thereof and compositionscontaining the same are useful in the treatment of conditions associatedwith modulating NO concentrations.

[0233] Example 9 described the antioxidant activity (as inhibitors offree radicals) of the inventive compounds. Given that NO is a freeradical and that the inventive compounds are strong antioxidants, it wassuspected that the administration of the inventive compounds toexperimental in vitro and in vivo models would have caused a reductionin NO levels. Any reduction in NO would have resulted in a hypertensive,rather than a hypotensive effect. Contrary to expectations, theinventive compounds elicited increases in NO from in vitro experimentsand produced a hypotensive effect from in vivo studies (Examples 31 and32). These results were unanticipated and completely unexpected.

[0234] Example 27 describes an erythmia (facial flush) shortly afterdrinking a solution containing the inventive compounds and glucose, thusimplying a vasodilation effect.

[0235] Example 31 describes the hypotensive effects elicited by theinventive compounds in an in vivo animal model, demonstrating theefficacy of the inventive compounds in the treatment of hypertension. Inthis example, the inventive compounds, combinations thereof andcompositions comprising the same comprise oligomers wherein n is 2 to18, and preferably, n is 2 to 10.

[0236] Example 32 describes the modulation of NO production by theinventive compounds in an in vitro model. In this example, the inventivecompounds, combinations thereof and compositions comprising the samecomprise oligomers wherein n is 2 to 18, and preferably n is 2 to 10.

[0237] Further, Example 35 provides evidence for the formation of Cu⁺²-,Fe⁺²- and Fe⁺³-oligomer complexes detected by MALDI/TOF/MS. Theseresults indicate that the inventive compounds can complex with copperand/or iron ions to minimize their effects on LDL oxidation.

[0238] Moreover, the inventive compounds have useful anti-microbialactivities for the treatment of infections and for the prevention offood spoilage. Examples 22 and 30 describe the antimicrobial activity ofthe inventive compounds against several representative microbiota havingclinical and food significance, as outlined below. CLINICAL/FOODMICROORGANISM TYPE RELEVANCE Helicobacter pylori gram negativegastritis, ulcers, gastric cancer Bacillus species gram positive foodpoisoning, wound infections, bovine mastitis, septicemia Salmonellaspecies gram negative food poisoning, diarrhea Staphylococcus grampositive boils, carbuncles, aureus wound infection, septicemia, breastabscesses Escherichia coli gram negative infant diarrhea, urinary tractinfection Pseudomonas species gram negative urinary tract infections,wound infections, “swimmer's ear” Saccharomyces yeast food spoilagecervisea Acetobacter gram negative food spoilage pasteurianus

[0239] Example 33 describes the effects of the inventive compounds onmacrophage NO production. In this example, the results demonstrate thatthe inventive compounds induce monocyte/macrophage NO production, bothindependent and dependent of stimulation by lipopolysaccharide (LPS) orcytokines. Macrophages producing NO can inhibit the growth of infectiousbacteria.

[0240] Compounds of the invention eliciting antimicrobial activity areoligomers, where n is 2 to 18, and preferably, are oligomers where n is2, 4, 5, 6, 8 and 10.

[0241] Examples of compounds eliciting the antimicrobial activity withrespect to NO cited above include dimers, tetramers, pentamers,hexamers, octamers and decamers, discussed above.

Anti-Cancer Utilities

[0242] Cancers are classified into three groups: carcinomas, sarcomasand lymphomas. A carcinoma is a malignancy that arises in the skin,linings of various organs, glands and tissues. A sarcoma is a malignancythat arises in the bone, muscle or connective tissue. The third groupcomprises leukemias and lymphomas because both develop within the bloodcell forming organs. The major types of cancer are prostate, breast,lung, colorectal, bladder, non-Hodgkin's lymphoma, uterine, melanoma ofthe skin, kidney, leukemia, ovarian and pancreatic.

[0243] The development of cancer results from alterations to the DNA ofcells which is brought about by many factors such as inheritable geneticfactors, ionizing radiation, pollutants, radon, and free radical damageto the DNA. Cells carrying mutations produce a defect in the orderedprocess of cell division. These cells fail to undergo apoptosis(programmed cell death) and continue to divide which either marks thebeginnings of a malignant tumor or allows more mutations to occur overtime to result in a malignancy.

[0244] There are three major features common to the many differentcancers. These are (1) the ability to proliferate indefinitely; (2)invasion of the tumor into the surrounding tissue; and (3) the processof metastasis.

[0245] Certain types of cancer metastasize in characteristic ways. Forexample, cancers of the thyroid gland, lung, breast, kidney and prostategland frequently metastasize to the bones. Lung cancer commonly spreadsto the brain and adrenal glands and colorectal cancer often metastasizesto the liver. Leukemia is considered to be a generalized disease at theonset, where it is found in the bone marrow throughout the body.

[0246] It has been surprisingly found that the inventive compounds areuseful in the treatment of a variety of cancers discussed above.Examples 6, 7, 8 and 15 describe the inventive compounds which elicitanti-cancer activity against human HeLa (cervical), prostate, breast,renal, T-cell leukemia and colon cancer cell lines. Example 12 (FIG. 20)illustrates the dose response effects on HeLa and SKBR-3 breast cancercell lines treated with oligomeric (dimers-dodecamers) procyanidins,which were substantially purified by HPLC. Cytotoxicity against thesecancer cell lines were dependent upon pentamer through dodecamerprocyanidins, with the lower oligomers showing no effect.

[0247] While not wishing to be bound by any theory, there appeared to bea minimum structural motif that accounts for the effects describedabove. Example 37 also shows the same cytotoxic effects of the higheroligomers (pentamer-decamer) against a feline lymphoblastoid cancer cellline. Cytotoxicity was also observed with higher oligomers (FIGS. 58 to61) against normal canine and feline cell lines.

[0248] In Example 8 (FIGS. 9D-H), the inventive compounds were shown toelicit cytotoxicity against a putative COX-2 expressing human coloncancer cell line (HCT 116).

[0249] Example 9 describes the antioxidant activity by the inventivecompounds. The compounds of the invention inhibit DNA strand breaks,DNA-protein cross-links and free radical oxidation of nucleotides toreduce and/or prevent the occurrence of mutations.

[0250] Example 10 describes the inventive compounds as topoisomerase IIinhibitors, which is a target for chemotherapeutic agents, such asdoxorubicin.

[0251] Example 21 describes the in vivo effects of a substantially purepentamer which elicited anti-tumor activity against a human breastcancer cell line (MDA-MB-231/LCC6) in a nude mouse model (average weightof a mouse is approximately 25 g). Repeat in vivo experiments with thepentamer at higher dosages (5 mg) have not entirely been successful, dueto unexpected animal toxicity. It is currently believed that thistoxicity may be related to the vasodilation effects of the inventivecompounds.

[0252] Example 33 describes the effects of the inventive compounds onmacrophage NO production. Macrophages which produce NO can inhibit thegrowth of rapidly dividing tumor cells.

[0253] Still further, the invention includes the use of the inventivecompounds to induce the inhibition of cellular proliferation byapoptosis.

[0254] For anti-cancer activity, the inventive compounds are oligomers,where n is 2 to 18, e.g., 3 to 18, such as 3 to 12, and preferably, n is5 to 12, and most preferably n is 5.

[0255] Compounds which elicit the inhibitory activity with respect tocancer cited above include pentamers to dodecamers, discussed above.

Formulations and Methods

[0256] Therefore, collectively, the inventive compounds, combinationsthereof and compositions comprising the same have exhibited a wide arrayof activities against several aspects of atherosclerosis, cardiovasculardisease, cancer, blood pressure modulation and/or hypertension,inflammatory disease, infectious agents and food spoilage.

[0257] Hence, the compounds of the invention, combinations thereof andcompositions containing the same are COX inhibitors which affectplatelet aggregation by inhibiting thromboxane A₂ formation, thusreducing the risk for thrombosis. Further, the inhibition of COX leadsto decreased platelet and inflammatory cell adhesion to vessel walls toallow for improved NO mechanisms of relaxation. These results, coupledwith the inhibition of COX at concentrations similar to a known NSAID,indomethacin, indicates antithrombotic efficacy.

[0258] Moreover, the compounds of the invention, combinations thereofand compositions containing the same are antioxidants which suppress theoxidation of LDL by reducing the levels of superoxide radical anion andlipoxygenase mediated lipid peroxy radicals. The inhibition of LDLoxidation at this stage slows macrophage activation and retards foamcell formation to interrupt further progression of atherosclerosis. Theinhibition of LDL oxidation can also slow the progression of restenosis.Thus, compounds of the invention or combinations thereof or compositionscontaining compounds of the invention or combinations thereof can beused for prevention and/or treatment of atherosclerosis and/orrestenosis. And thus, the inventive compounds can be administered beforeor after angioplasty or similar procedures to prevent or treatrestenosis in patients susceptible thereto.

[0259] For treatment or prevention of restenosis and/or atherosclerosis,an inventive compound or compounds or a composition comprising aninventive compound or compounds, alone or with other treatment, may beadministered as desired by the skilled medical practitioner, from thisdisclosure and knowledge in the art, e.g., at the first signs orsymptoms of restenosis and/or atherosclerosis, immediately prior to,concomitant with or after angioplasty, or as soon thereafter as desiredby the skilled medical practitioner, without any undue experimentationrequired; and the administration of the inventive compound or compoundsor a composition thereof, alone or with other treatment, may becontinued as a regimen, e.g., monthly, bimonthly, biannually, annually,or in some other regimen, by the skilled medical practitioner for suchtime as is necessary, without any undue experimentation required.

[0260] Further, the compounds of the invention, combinations thereof andcompositions comprising the same have been shown to produce ahypotensive effect in vivo and induce NO in vitro. These results havepractical application in the treatment of hypertension and in clinicalsituations involving hypercholesterolemia, where NO levels are markedlyreduced.

[0261] Formulations of the inventive compounds, combinations thereof andcompositions comprising the same can be prepared with standardtechniques well known to those skilled in the pharmaceutical, foodscience, medical and veterinary arts, in the form of a liquid,suspension, tablet, capsule, injectable solution or suppository, forimmediate or slow-release of the active compounds.

[0262] The carrier may also be a polymeric delayed release system.Synthetic polymers are particularly useful in the formulation of acomposition having controlled release. An early example of this was thepolymerization of methyl methacrylate into spheres having diameters lessthan one micron to form so-called nano particles, reported by Kreuter,J., Microcapsules and Nanoparticles in Medicine and Pharmacology, M.Donbrow (Ed). CRC Press, p. 125-148.

[0263] A frequent choice of a carrier for pharmaceuticals and morerecently for antigens is poly (d,1-lactide-co-glycolide) (PLGA). This isa biodegradable polyester that has a long history of medical use inerodible sutures, bone plates and other temporary prostheses where ithas not exhibited any toxicity. A wide variety of pharmaceuticals havebeen formulated into PLGA microcapsules. A body of data has accumulatedon the adaption of PLGA for controlled, for example, as reviewed byEldridge, J. H., et al. Current Topics in Microbiology and Immunology,1989, 146:59-66. The entrapment in PLGA microspheres of 1 to 10 micronsin diameter can have an effect when administered orally. The PLGAmicroencapsulation process uses a phase separation of a water-in-oilemulsion. The inventive compound or compounds is or are prepared as anaqueous solution and the PLGA is dissolved in a suitable organicsolvents such as methylene chloride and ethyl acetate. These twoimmiscible solutions are co-emulsified by high-speed stirring. Anon-solvent for the polymer is then added, causing precipitation of thepolymer around the aqueous droplets to form embryonic microcapsules. Themicrocapsules are collected, and stabilized with one of an assortment ofagents (polyvinyl alcohol (PVA), gelatin, alginates, methyl cellulose)and the solvent removed by either drying in vacuo or solvent extraction.

[0264] Additionally, with regard to the preparation of slow-releaseformulations, reference is made to U.S. Pat. Nos. 5,024,843, 5,091,190,5,082,668, 4,612,008 and 4,327,725, hereby incorporated herein byreference.

[0265] Additionally, selective processing coupled with theidentification of cocoa genotypes of interest could be used to prepareStandard-of-Identity (SOI) and non-SOI chocolate products as vehicles todeliver the active compounds to a patient in need of treatment for thedisease conditions described above, as well as a means for the deliveryof conserved levels of the inventive compounds.

[0266] In this regard, reference is made to copending U.S. applicationSer. No. 08/709,406, filed Sep. 6, 1996, hereby incorporated herein byreference. U.S. Ser. No. 08/709,406 relates to a method of producingcocoa butter and/or cocoa solids having conserved levels of polyphenolsfrom cocoa beans using a unique combination of processing steps whichdoes not require separate bean roasting or liquor milling equipment,allowing for the option of processing cocoa beans without exposure tosevere thermal treatment for extended periods of time and/or the use ofsolvent extraction of fat. The benefit of this process lies in theenhanced conservation of polyphenols in contrast to that found intraditional cocoa processing, such that the ratio of the initial amountof polyphenol found in the unprocessed bean to that obtainable afterprocessing is less than or equal to 2.

[0267] Compositions of the invention include one or more of the abovenoted compounds in a formulation having a pharmaceutically acceptablecarrier or excipient, the inventive compounds having anti-cancer,anti-tumor or antineoplastic activities, antioxidant activity, inhibitDNA topoisomeriase II enzyme, inhibit oxidative damage to DNA, inducemonocyte/macrophage NO production, have antimicrobial, cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, apoptosis, platelet aggregationand blood or in vivo glucose modulating activities, and have efficacy asnon-steroidal antiinflammatory agents.

[0268] Another embodiment of the invention includes compositionscomprising the inventive compounds or combinations thereof, as well asat least one additional antineoplastic, blood pressure reducing,antiinflammatory, antimicrobial, antioxidant and hematopoiesis agents,in addition to a pharmaceutically acceptable carrier or excipient.

[0269] Such compositions can be administered to a subject or patient inneed of such administration in dosages and by techniques well known tothose skilled in the medical, nutritional or veterinary arts taking intoconsideration the data herein, and such factors as the age, sex, weight,genetics and condition of the particular subject or patient, and theroute of administration, relative concentration of particular oligomers,and toxicity (e.g., LD₅₀).

[0270] The compositions can be co-administered or sequentiallyadministered with other antineoplastic, anti-tumor or anti-canceragents, antioxidants, DNA topoisomerase II enzyme inhibiting agents,inhibitors of oxidatively damaged DNA or cyclo-oxygenase and/orlipoxygenase, apoptosis, platelet aggregation, blood or in vivo glucoseor NO or NO-synthase modulating agents, non-steroidal antiinflammatoryagents and/or with agents which reduce or alleviate ill effects ofantineoplastic, anti-tumor, anti-cancer agents, antioxidants, DNAtopoisomerase II enzyme inhibiting agents, inhibitors of oxidativelydamaged DNA, cyclo-oxygenase and/or lipoxygenase, apoptosis, plateletaggregation, blood or in vivo glucose or NO or NO-synthase modulatingand/or non-steroidal antiinflammatory agents; again, taking intoconsideration such factors as the age, sex, weight, genetics andcondition of the particular subject or patient, and, the route ofadministration.

[0271] Examples of compositions of the invention for human or veterinaryuse include edible compositions for oral administration, such solid orliquid formulations, for instance, capsules, tablets, pills and thelike, as well as chewable solid or beverage formulations, to which thepresent invention may be well-suited since it is from an edible source(e.g., cocoa or chocolate flavored solid or liquid compositions); liquidpreparations for orifice, e.g., oral, nasal, anal, vaginal etc.,administration such as suspensions, syrups or elixirs (including cocoaor chocolate flavored compositions); and, preparations for parental,subcutaneous, intradermal, intramuscular or intravenous administration(e.g., injectable administration) such as sterile suspensions oremulsions. However, the active ingredient in the compositions maycomplex with proteins such that when administered into the bloodstream,clotting may occur due to precipitation of blood proteins; and, theskilled artisan should take this into account. In such compositions theactive cocoa extract may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, DMSO, ethanol, or the like. The active cocoa extract of theinvention can be provided in lyophilized form for reconstituting, forinstance, in isotonic aqueous, saline, glucose or DMSO buffer. Incertain saline solutions, some precipitation has been observed; and,this observation may be employed as a means to isolate inventivecompounds, e.g., by a “salting out” procedure.

[0272] Example 38 describes the preparation of the inventive compoundsin a tablet formulation for application in the pharmaceutical,supplement and food areas. Further, Example 39 describes the preparationof the inventive compounds in capsule formulations for similarapplications. Still further, Example 40 describes the formulation ofStandard of Identity (SOI) and non-SOI chocolates containing thecompounds of the invention or cocoa solids obtained from methodsdescribed in copending U.S. application Ser. No. 08/709,406, herebyincorporated herein by reference.

Kits

[0273] Further, the invention also comprehends a kit wherein the activecocoa extract is provided. The kit can include a separate containercontaining a suitable carrier, diluent or excipient. The kit can alsoinclude an additional anti-cancer, anti-tumor or antineoplastic agent,antioxidant, DNA topoisomerase II enzyme inhibitor or an inhibitor ofoxidative DNA damage or antimicrobial, or cyclo-oxygenase and/orlipoxygenase, NO or NO-synthase non-steroidal antiinflammatory,apoptosis and platelet aggregation modulating or blood or in vivoglucose modulating agent and/or an agent which reduces or alleviates illeffects of antineoplastic, anti-tumor or anti-cancer agents,antioxidant, DNA topoisomerase II enzyme inhibitor or antimicrobial, orcyclo-oxygenase and/or lipoxygenase, NO or NO-synthase, apoptosis,platelet aggregation and blood or in vivo glucose modulating and/ornon-steroidal antiinflammatory agents for co- orsequential-administration. The additional agent(s) can be provided inseparate container(s) or in admixture with the active cocoa extract.Additionally, the kit can include instructions for mixing or combiningingredients and/or administration.

Identification of Genes

[0274] A further embodiment of the invention comprehends the modulationof genes expressed as a result of intimate cellular contact by theinventive compounds or a combination of compounds. As such, the presentinvention comprehends methods for the identification of genes induced orrepressed by the inventive compounds or a combination of compounds whichare associated with several diseases, including but not limited toatherosclerosis, hypertension, cancer, cardiovascular disease, andinflammation. Specifically, genes which are differentially expressed inthese disease states, relative to their expression in “normal”nondisease states are identified and described before and after contactby the inventive compounds or a combination of compounds.

[0275] As mentioned in the previous discussion, these diseases anddisease states are based in part on free radical interactions with adiversity of biomolecules. A central theme in these diseases is thatmany of the free radical reactions involve reactive oxygen species,which in turn induce physiological conditions involved in diseaseprogression. For instance, reactive oxygen species have been implicatedin the regulation of transcription factors such as nuclear factor(NF)-κB. The target genes for NF-κB comprise a list of genes linked tocoordinated inflammatory response. These include genes encoding tumornecrosis factor (TNF)-α, interleukin (IL)-I, IL-6, IL-8, inducible NOS,Major Histocompatabilty Complex (MHC) class I antigens, and others.Also, genes that modulate the activity of transcription factors may inturn be induced by oxidative stress. Oxidative stress is the imbalancebetween radical scavenging and radical generating systems. Several knownexamples (Winyard and Blake, 1997) of these conditions include gadd153(a gene induced by growth arrest and DNA damage), the product of whichhas been shown to bind NF-IL6 and form a heterodimer that cannot bind toDNA. NF-IL6 upregulates the expression of several genes, including thoseencoding interleukins 6 and 8. Another example of oxidative stressinducible genes are gadd45 which regulates the effects of thetranscription factor p53 in growth arrest. p53 codes for the p53 proteinwhich can halt cell division and induce abnormal cells (e.g. cancer) toundergo apoptosis.

[0276] Given the full panoply of unexpected, nonobvious and novelutilities for the inventive compounds or combination of compounds forutility in a diverse array of diseases based in part by free radicalmechanisms, the invention further comprehends strategies to determinethe temporal effects on gene(s) or gene product(s) expression by theinventive compounds in animal in vitro and/or in vivo models of specificdisease or disease states using gene expression assays. These assaysinclude, but are not limited to Differential Display, sequencing of cDNAlibraries, Serial Analysis of Gene Expression (SAGE), expressionmonitoring by hybridization to high density oligonucleotide arrays andvarious reverse transcriptase-polymerization chain reaction (RT-PCR)based protocols or their combinations (Lockhart et al., 1996).

[0277] The comprehensive physiological effects of the inventivecompounds or combination of compounds embodied in the invention, coupledto a genetic evaluation process permits the discovery of genes and geneproducts, whether known or novel, induced or repressed. For instance,the invention comprehends the in vitro and in vivo induction and/orrepression of cytokines (e.g. IL-1, IL-2, IL-6, IL-8, IL-12, and TNF-α)in lymphocytes using RT-PCR. Similarly, the invention comprehends theapplication of Differential Display to ascertain the induction and/orrepression of select genes; for the cardiovascular area (e.g. superoxidedismutase, heme oxidase, COX I and 2, and other oxidant defense genes)under stimulated and/or oxidant stimulated conditions (e.g. TNF-α orH₂O₂) conditions. For the cancer area, the invention comprehends theapplication of Differential Display to ascertain the induction and/orrepression of genes or gene products such as CuZn-superoxide dismutase,Mn-superoxide dismutase, catalase, etc., in control and oxidant stressedcells.

[0278] The following non-limiting Examples are given by way ofillustration only and are not to be considered a limitation of thisinvention, many apparent variations of which are possible withoutdeparting from the spirit or scope thereof.

EXAMPLES Example 1 Cocoa Source and Method of Preparation

[0279] Several Theobroma cacao genotypes which represent the threerecognized horticultural races of cocoa (Enriquez, 1967; Engels, 1981)were obtained from the three major cocoa producing origins of the world.A list of those genotypes used in this study are shown in Table 1.Harvested cocoa pods were opened and the beans with pulp were removedfor freeze drying. The pulp was manually removed from the freeze driedmass and the beans were subjected to analysis as follows. Theunfermented, freeze dried cocoa beans were first manually dehulled, andground to a fine powdery mass with a TEKMAR Mill. The resultant mass wasthen defatted overnight by Soxhlet extraction using redistilled hexaneas the solvent. Residual solvent was removed from the defatted mass byvacuum at ambient temperature. TABLE 1 Description of Theobroma cacaoSource Material GENOTYPE ORIGIN HORTICULTURAL RACE UIT-1 MalaysiaTrinitario Unknown West Africa Forastero ICS-100 Brazil Trinitario(Nicaraguan Criollo ancestor) ICS-39 Brazil Trinitario (NicaraguanCriollo ancestor) UF-613 Brazil Trinitario EEG-48 Brazil Forastero UF-12Brazil Trinitario NA-33 Brazil Forastero

Example 2 Procyanidin Extraction Procedures

[0280] A. Method 1

[0281] Procyanidins were extracted from the defatted, unfermented,freeze dried cocoa beans of Example 1 using a modification of the methoddescribed by Jalal and Collin (1977). Procyanidins were extracted from50 gram batches of the defatted cocoa mass with 2×400 mL 70%acetone/deionized water followed by 400 mL 70% methanol/deionized water.The extracts were pooled and the solvents removed by evaporation at 45°C. with a rotary evaporator held under partial vacuum. The resultantaqueous phase was diluted to 1 L with deionized water and extracted 2×with 400 mL CHCl₃. The solvent phase was discarded. The aqueous phasewas then extracted 4× with 500 mL ethyl acetate. Any resultant emulsionswere broken by centrifugation on a Sorvall RC 28S centrifuge operated at2,000×g for 30 min. at 10° C. To the combined ethyl acetate extracts,100-200 mL deionized water was added. The solvent was removed byevaporation at 45° C. with a rotary evaporator held under partialvacuum. The resultant aqueous phase was frozen in liquid N₂ followed byfreeze drying on a LABCONCO Freeze Dry System. The yields of crudeprocyanidins that were obtained from the different cocoa genotypes arelisted in Table 2. TABLE 2 Crude Procyanidin Yields GENOTYPE ORIGINYIELDS (g) UIT-1 Malaysia 3.81 Unknown West Africa 2.55 ICS-100 Brazil3.42 ICS-39 Brazil 3.45 UF-613 Brazil 2.98 EEG-48 Brazil 3.15 UF-12Brazil 1.21 NA-33 Brazil 2.23

[0282] B. Method 2

[0283] Alternatively, procyanidins are extracted from defatted,unfermented, freeze dried cocoa beans of Example 1 with 70% aqueousacetone. Ten grams of defatted material was slurried with 100 mL solventfor 5-10 min. The slurry was centrifuged for 15 min. at 4° C. at 3000×gand the supernatant passed through glass wool. The filtrate wassubjected to distillation under partial vacuum and the resultant aqueousphase frozen in liquid N₂, followed by freeze drying on a LABCONCOFreeze Dry System. The yields of crude procyanidins ranged from 15-20%.

[0284] Without wishing to be bound by any particular theory, it isbelieved that the differences in crude yields reflected variationsencountered with different genotypes, geographical origin, horticulturalrace, and method of preparation.

Example 3 Partial Purification of Cocoa Procyanidins

[0285] A. Gel Permeation Chromatography

[0286] Procyanidins obtained from Example 2 were partially purified byliquid chromatography on Sephadex LH-20 (28×2.5 cm). Separations wereaided by a step gradient from deionized water into methanol. The initialgradient composition started with 15% methanol in deionized water whichwas followed step wise every 30 min. with 25% methanol in deionizedwater, 35% methanol in deionized water, 70% methanol in deionized water,and finally 100% methanol. The effluent following the elution of thexanthine alkaloids (caffeine and theobromine) was collected as a singlefraction. The fraction yielded a xanthine alkaloid free subfractionwhich was submitted to further subfractionation to yield fivesubfractions designated MM2A through MM2E. The solvent was removed fromeach subfraction by evaporation at 45° C. with a rotary evaporator heldunder partial vacuum. The resultant aqueous phase was frozen in liquidN₂ and freeze dried overnight on a LABCONCO Freeze Dry System. Arepresentative gel permeation chromatogram showing the fractionation isshown in FIG. 1. Approximately, 100 mg of material was subfractionatedin this manner.

[0287] Chromatographic Conditions: Column; 28×2.5 cm Sephadex LH-20,Mobile Phase: Methanol/Water Step Gradient, 15:85, 25:75, 35:65, 70:30,100:0 Stepped at ½ Hour Intervals, Flow Rate; 1.5 mL/min, Detector; UVat λ₁=254 nm and λ₂=365 nm, Chart Speed: 0.5 mm/min, Column Load; 120mg.

[0288] B. Semi-Preparative High Performance Liquid Chromatography (HPLC)

[0289] Method 1. Reverse Phase Separation

[0290] Procyanidins obtained from Example 2 and/or 3A were partiallypurified by semi-preparative HPLC. A Hewlett Packard 1050 HPLC Systemequipped with a variable wavelength detector, Rheodyne 7010 injectionvalve with 1 mL injection loop was assembled with a Pharmacia FRAC-100Fraction Collector. Separations were effected on a Phenomenex Ultracarb™10μ ODS column (250×22.5 mm) connected with a Phenomenex 10μ ODSUltracarb™ (60×10 mm) guard column. The mobile phase composition wasA=water; B=methanol used under the following linear gradient conditions:[Time, %A]; (0, 85), (60, 50), (90, 0), and (110, 0) at a flow rate of 5mL/min. Compounds were detected by UV at 254 nm

[0291] A representative Semi-preparative HPLC trace is shown in FIG. 15Nfor the separation of procyanidins present in fraction D+E. Individualpeaks or select chromatographic regions were collected on timedintervals or manually by fraction collection for further purificationand subsequent evaluation. Injection loads ranged from 25-100 mg ofmaterial.

[0292] Method 2. Normal Phase Separation

[0293] Procyanidin extracts obtained from Examples 2 and/or 3A werepartially purified by semi-preparative HPLC. A Hewlett Packard 1050 HPLCsystem, Millipore-Waters Model 480 LC detector set at 254 nm wasassembled with a Pharmacia Frac-100 Fraction Collector set in peak mode.Separations were effected on a Supelco 5 μm Supelcosil LC-Si column(250×10 mm) connected with a Supelco 5 μm Supelguard LC-Si guard column(20×4.6 mm). Procyanidins were eluted by a linear gradient under thefollowing conditions: (Time, %A, %B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10 min.re-equilibration. Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). A flow rate of 3 mL/min wasused. Components were detected by UV at 254 nm, and recorded on a Kipp &Zonan BD41 recorder. Injection volumes ranged from 100-250 μL of 10 mgof procyanidin extracts dissolved in 0.25 mL 70% aqueous acetone. Arepresentative semi-preparative HPLC trace is shown in FIG. 15 O.Individual peaks or select chromatographic regions were collected ontimed intervals or manually by fraction collection for furtherpurification and subsequent evaluation. HPLC Conditions: 250 × 10 mmSupelco Supelcosil LC-Si (5 μm) Semipreparative Column 20 × 4.6 mmSupelco Supelcosil LC-Si (5 μm) Guard Column Detector: Waters LCSpectrophotometer Model 480 @ 254 nm Flow rate: 3 mL/min, ColumnTemperature: ambient, Injection: 250 μL of 70% aqueous acetone extract.Gradient: Acetic Time (min) CH₂Cl₂ Methanol Acid:H₂O (1:1)  0 82 14 4 3067.6 28.4 4 60 46 50 4 65 10 86 4 70 10 86 4

[0294] The fractions obtained were as follows: FRACTION TYPE 1 dimers 2trimers 3 tetramers 4 pentamers 5 hexamers 6 heptamers 7 octamers 8nonamers 9 decamers 10  undecamers 11  dodecamers 12  higher oligomers

Example 4 Analytical HPLC Analysis of Procyanidin Extracts

[0295] Method 1. Reverse Phase Separation

[0296] Procyanidin extracts obtained from Example 3 were filteredthrough a 0.45μ filter and analyzed by a Hewlett Packard 1090 ternaryHPLC system equipped with a Diode Array detector and a HP model 1046AProgrammable Fluorescence Detector. Separations were effected at 45° C.on a Hewlett-Packard 5μ Hypersil ODS column (200×2.1 mm). The flavanolsand procyanidins were eluted with a linear gradient of 60% B into Afollowed by a column wash with B at a flow rate of 0.3 mL/min. Themobile phase composition was B=0.5% acetic acid in methanol and A=0.5%acetic acid in nanopure water. Acetic acid levels in A and B mobilephases can be increased to 2%. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(ex)=316 nm and by UV at 280 nm.Concentrations of (+)-catechin and (−)-epicatechin were determinedrelative to reference standard solutions. Procyanidin levels wereestimated by using the response factor for (−)-epicatechin. Arepresentative HPLC chromatogram showing the separation of the variouscomponents is shown in FIG. 2A for one cocoa genotype. Similar HPLCprofiles were obtained from the other cocoa genotypes. HPLC Conditions:Column: 200 × 2.1 mm Hewlett Packard Hypersil ODS (5 μ) Guard column: 20× 2.1 mm Hewlett Packard Hypersil ODS (5 μ) Detectors: Diode Array @ 280mm Fluorescence λ_(ex) = 276 nm; μ_(em) = 316 nm. Flow rate: 0.3 mL/min.Column Temperature: 45° C. Gradient: 0.5% Acetic Acid 0.5% Acetic acidTime (min) in nanopure water in methanol  0 100  0 50  40  60 60  0 100

[0297] Method 2. Normal Phase Separation

[0298] Procyanidin extracts obtained from Examples 2 and/or 3 werefiltered through a 0.45μ filter and analyzed by a Hewlett Packard 1090Series II HPLC system equipped with a HP model 1046A ProgrammableFluorescence detector and Diode Array detector. Separations wereeffected at 37° C. on a 5μ Phenomenex Lichrosphere® Silica 100 column(250×3.2 mm) connected to a Supelco Supelguard LC-Si 5μ guard column(20×4.6 mm). Procyanidins were eluted by linear gradient under thefollowing conditions: (Time, %A, %B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (70, 10, 86) followed by an 8 min.re-equilibration. Mobile phase composition was A=dichloromethane,B=methanol, and C=acetic acid: water at a volume ratio of 1:1. A flowrate of 0.5 mL/min. was used. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(em)=316 nm or by UV at 280 nm. Arepresentative HPLC chromatogram showing the separation of the variousprocyanidins is shown in FIG. 2B for one genotype. Similar HPLC profileswere obtained from other cocoa genotypes. HPLC Conditions: 250 × 3.2 mmPhenomenex Lichrosphere ® Silica 100 column (5 μ) 20 × 4.6 mm SupelcoSupelguard LC-Si (5 μ) guard column Detectors: Photodiode Array @ 280 nmFluorescence λ_(ex) = 276 nm; λ_(em) = 316 nm. Flow rate: 0.5 mL/min.Column Temperature: 37° C. Acetic Gradient: Acid/Water Time (min.)CH₂—Cl₂ Methanol (1:1)  0 82 14 4 30 67.6 28.4 4 60 46 50 4 65 10 86 470 10 86 4

Example 5 Identification of Procyanidins

[0299] Procyanidins were purified by liquid chromatography on SephadexLH-20 (28×2.5 cm) columns followed by semi-preparative HPLC using a 10μBondapak C18 (100×8 mm) column or by semi-preparative HPLC using a 5μSupelcosil LC-Si (250×10 mm) column.

[0300] Partially purified isolates were analyzed by Fast AtomBombardment-Mass Spectrometry (FAB-MS) on a VG ZAB-T high resolution MSsystem using a Liquid Secondary Ion Mass Spectrometry (LSIMS) techniquein positive and negative ion modes. A cesium ion gun was used as theionizing source at 30 kV and a “Magic Bullet Matrix” (1:1dithiothreitol/dithioerythritol) was used as the proton donor.

[0301] Analytical investigations of these fractions by LSIMS revealedthe presence of a number of flavan-3-ol oligomers as shown in TableTABLE 3 LSIMS (Positive Ion) Data from Cocoa Procyanidin Fractions (M +1)⁺ (M + Na)⁺ Oligoiner m/z m/z Mol. Wt. Monomers  291  313  290(catechins) Dimer(s) 577/579 599/601 576/578 Trimer(s) 865/867 887/889864/866 Tetramer(s) 1155 1177 1154 Pentamer(s) 1443 1465 1442 Hexamer(s)1731 1753 1730 Heptamer(s) — 2041 2018 Octamer(s) — 2329 2306 Nonamer(s)— 2617 2594 Decamer(s) — 2905 2882 Undecamer(s) — — 3170 Dodecamer(s) —— 3458

[0302] The major mass fragment ions were consistent with work previouslyreported for both positive and negative ion FAB-MS analysis ofprocyanidins (Self et al., 1986 and Porter et al., 1991). The ioncorresponding to m/z 577 (M+H)⁺ and its sodium adduct at m/z 599 (M+Na)⁺suggested the presence of doubly linked procyanidin dimers in theisolates. It was interesting to note that the higher oligomers were morelikely to form sodium adducts (M+Na)⁺ than their protonated molecularions (M+H)⁺. The procyanidin isomers B-2, B-5 and C-1 were tentativelyidentified based on the work reported by Revilla et al. (1991), Self etal. (1986) and Porter et al. (1991). Procyanidins up to both the octamerand decamer were verified by FAB-MS in the partially purified fractions.Additionally, evidence for procyanidins up to the dodecamer wereobserved from normal phase HPLC analysis (see FIG. 2B). Table 4 liststhe relative concentrations of the procyanidins found in xanthinealkaloid free isolates based on reverse phase HPLC analysis. Table 5lists the relative concentrations of the procyanidins based on normalphase HPLC analysis. TABLE 4 Relative Concentrations of Procyanidins inthe Xanthine Alkaloid Free Isolates Component Amount (+)-catechin 1.6%(−)-epicatechin 38.2%  B-2 Dimer 11.0%  B-5 Dimer 5.3% C-1 Trimer 9.3%Doubly linked 3.0% dimers Tetramer(s) 4.5% Pentamer-Octamer 24.5% Unknowns and 2.6% higher oligoiners

[0303] TABLE 5 Relative Concentrations of Procyanidins in AqueousAcetone Extracts Component Amount (+)-catechin and 41.9% (−)-epicatechin B-2 and B-5 Dimers 13.9%  Trimers 11.3%  Tetramers 9.9%Pentamers 7.8% Hexamers 5.1% Heptamers 4.2% Octamers 2.8% Nonamers 1.6%Decamers 0.7% Undecamers 0.2% Dodecamers <0.1%  

[0304]FIG. 3 shows several procyanidin structures and FIGS. 4A-4E showthe representative HPLC chromatograms of the five fractions employed inthe following screening for anti-cancer or antineoplastic activity. TheHPLC conditions for FIGS. 4A-4E were as follows:

[0305] HPLC Conditions: Hewlett Packard 1090 ternary HPLC Systemequipped with HP Model 1046A Programmable Fluorescence Detector.

[0306] Column: Hewlett Packard 5μ Hypersil ODS (200×2.1 mm) LinearGradient of 60% B into A at a flow rate of 0.3 mL/min. B=0.5% aceticacid in methanol; A=0.5% acetic acid in deionized water. λ_(ex)=280 nm;λ_(em)=316 nm.

[0307]FIG. 15 O shows a representative semi-prep HPLC chromatogram of anadditional 12 fractions employed in the screening for anticancer orantineoplastic activity (HPLC conditions stated above).

Example 6 Anti-Cancer, Anti-Tumor or Antineoplastic Activity of CocoaExtracts (Procyanidins)

[0308] The MTT (3-[4,5-dimethyl thiazol-2yl]-2,5-diphenyltetrazoliumbromide)-microtiter plate tetrazolium cytotoxicity assay originallydeveloped by Mosmann (1983) was used to screen test samples from Example5. Test samples, standards (cisplatin and chlorambucil) and MTT reagentwere dissolved in 100% DMSO (dimethyl sulfoxide) at a 10 mg/mLconcentration. Serial dilutions were prepared from the stock solutions.In the case of the test samples, dilutions ranging from 0.01 through 100μg/mL were prepared in 0.5% DMSO.

[0309] All human tumor cell lines were obtained from the American TypeCulture Collection. Cells were grown as mono layers in alpha-MEMcontaining 10% fetal bovine serum, 100 units/mL penicillin, 100 μg/mLstreptomycin and 240 units/mL nystatin. The cells were maintained in ahumidified, 5% CO₂ atmosphere at 37° C.

[0310] After trypsinization, the cells are counted and adjusted to aconcentration of 50×10⁵ cells/mL (varied according to cancer cell line).200 μL of the cell suspension was plated into wells of 4 rows of a96-well microtiter plate. After the cells were allowed to attach forfour hours, 2 μL of DMSO containing test sample solutions were added toquadruplicate wells. Initial dose-response finding experiments, usingorder of magnitude test sample dilutions were used to determine therange of doses to be examined. Well absorbencies at 540 nm were thenmeasured on a BIO RAD MP450 plate reader. The mean absorbance ofquadruplicate test sample treated wells was compared to the control, andthe results expressed as the percentage of control absorbance plus/minusthe standard deviation. The reduction of MTT to a purple formazanproduct correlates in a linear manner with the number of living cells inthe well. Thus, by measuring the absorbance of the reduction product, aquantitation of the percent of cell survival at a given dose of testsample can be obtained. Control wells contained a final concentration of1% DMSO.

[0311] Two of the samples were first tested by this protocol. Sample MM1represented a very crude isolate of cocoa procyanidins and containedappreciable quantities of caffeine and theobromine. Sample MM2represented a cocoa procyanidin isolate partially purified by gelpermeation chromatography. Caffeine and theobromine were absent in MM2.Both samples were screened for activity against the following cancercell lines using the procedures previously described:

[0312] HCT 116 colon cancer

[0313] ACHN renal adenocarcinoma

[0314] SK-5 melanoma

[0315] A498 renal adenocarcinoma

[0316] MCF-7 breast cancer

[0317] PC-3 prostate cancer

[0318] CAPAN-2 pancreatic cancer

[0319] Little or no activity was observed with MM1 on any of the cancercell lines investigated. MM2 was found to have activity against HCT-116,PC-3 and ACHN cancer cell lines. However, both MM1 and MM2 were found tointerfere with MTT such that it obscured the decrease in absorbance thatwould have reflected a decrease in viable cell number. This interferencealso contributed to large error bars, because the chemical reactionappeared to go more quickly in the wells along the perimeter of theplate. A typical example of these effects is shown in FIG. 5. At thehigh concentrations of test material, one would have expected to observea large decrease in survivors rather than the high survivor levelsshown. Nevertheless, microscopic examinations revealed that cytotoxiceffects occurred, despite the MTT interference effects. For instance, anIC₅₀ value of 0.5 μg/mL for the effect of MM2 on the ACHN cell line wasobtained in this manner.

[0320] These preliminary results, in the inventors' view, requiredamendment of the assay procedures to preclude the interference with MTT.This was accomplished as follows. After incubation of the plates at 37°C. in a humidified, 5% CO₂ atmosphere for 18 hours, the medium wascarefully aspirated and replaced with fresh alpha-MEM media. This mediawas again aspirated from the wells on the third day of the assay andreplaced with 100 μL of freshly prepared McCoy's medium. 11 μL of a 5mg/mL stock solution of MTT in PBS (Phosphate Buffered Saline) were thenadded to the wells of each plate. After incubation for 4 hours in ahumidified, 5% CO₂ atmosphere at 37° C., 100 μL of 0.04 N HCl inisopropanol was added to all wells of the plate, followed by thoroughmixing to solubilize the formazan produced by any viable cells.Additionally, it was decided to subfractionate the procyanidins todetermine the specific components responsible for activity.

[0321] The subfractionation procedures previously described were used toprepare samples for further screening. Five fractions representing theareas shown in FIG. 1 and component(s) distribution shown in FIGS. 4A-4Ewere prepared. The samples were coded MM2A through MM2E to reflect theseanalytical characterizations and to designate the absence of caffeineand theobromine.

[0322] Each fraction was individually screened against the HCT-116, PC-3and ACHN cancer cell lines. The results indicated that the activity didnot concentrate to any one specific fraction. This type of result wasnot considered unusual, since the components in “active” natural productisolates can behave synergistically. In the case of the cocoaprocyanidin isolate (MM2), over twenty detectable components comprisedthe isolate. It was considered possible that the activity was related toa combination of components present in the different fractions, ratherthan the activity being related to an individual component(s).

[0323] On the basis of these results, it was decided to combine thefractions and repeat the assays against the same cancer cell lines.Several fraction combinations produced cytotoxic effects against thePC-3 cancer cell lines. Specifically, IC₅₀ values of 40 μg/mL each forMM2A and MM2E combination, and of 20 μg/mL each for MM2C and MM2Ecombination, were obtained. Activity was also reported against theHCT-116 and ACHN cell lines, but as before, interference with the MTTindicator precluded precise observations. Replicate experiments wererepeatedly performed on the HCT-116 and ACHN lines to improve the data.However, these results were inconclusive due to bacterial contaminationand exhaustion of the test sample material. FIGS. 6A-6D show thedose-response relationship between combinations of the cocoa extractsand PC-3 cancer cells.

[0324] Nonetheless, from this data, it is clear that cocoa extracts,especially cocoa polyphenols or procyanidins, have significantanti-tumor; anti-cancer or antineoplastic activity, especially withrespect to human PC-3 (prostate), HCT-116 (colon) and ACHN (renal)cancer cell lines. In addition, those results suggest that specificprocyanidin fractions may be responsible for the activity against thePC-3 cell line.

Example 7 Anti-Cancer, Anti-Tumor or Antineoplastic Activity of CocoaExtracts (Procyanidins)

[0325] To confirm the above findings and further study fractioncombinations, another comprehensive screening was performed.

[0326] All prepared materials and procedures were identical to thosereported above, except that the standard 4-replicates per test dose wasincreased to 8 or 12-replicates per test dose. For this study,individual and combinations of five cocoa procyanidin fractions werescreened against the following cancer cell lines.

[0327] PC-3 Prostate

[0328] KB Nasopharyngeal/HeLa

[0329] HCT-116 Colon

[0330] ACHN Renal

[0331] MCF-7 Breast

[0332] SK-5 Melanoma

[0333] A-549 Lung

[0334] CCRF-CEM T-cell leukemia

[0335] Individual screenings consisted of assaying different dose levels(0.01-100 μg/mL) of fractions A, B, C, D, and E (See FIGS. 4A-4E anddiscussion thereof, supra) against each cell line. Combinationscreenings consisted of combining equal dose levels of fractions A+B,A+C, A+D, A+E, B+C, B+D, B+E, C+D, C+E, and D+E against each cell line.The results from these assays are individually discussed, followed by anoverall summary.

[0336] A. PC-3 Prostate Cell Line

[0337] FIGS. 7A-7H show the typical dose response relationship betweencocoa procyanidin fractions and the PC-3 cell line. FIGS. 7D and 7Edemonstrate that fractions D and E were active at an IC₅₀ value of 75μg/mL. The IC₅₀ values that were obtained from dose-response curves ofthe other procyanidin fraction combinations ranged between 60-80 μg/mLwhen fractions D or E were present. The individual IC₅₀ values arelisted in Table 6.

[0338] B. KB Nasopharyngeal/HeLa Cell Line

[0339] FIGS. 8A-8H show the typical dose response relationship betweencocoa procyanidin fractions and the KB Nasopharyngeal/HeLa cell line.FIGS. 8D and 8E demonstrate that fractions D and E were active at anIC₅₀ value of 75 μg/mL. FIGS. 8F-8H depict representative resultsobtained from the fraction combination study. In this case, procyanidinfraction combination A+B had no effect, whereas fraction combinationsB+E and D+E were active at an IC₅₀ value of 60 μg/mL. The IC₅₀ valuesthat were obtained from other dose response curves from other fractioncombinations ranged from 60-80 μg/mL when fractions D or E were present.The individual IC₅₀ values are listed in Table 6. These results wereessentially the same as those obtained against the PC-3 cell line.

[0340] C. HCT-116 Colon Cell Line

[0341]FIG. 9A-9H show the typical dose response relationships betweencocoa procyanidin fractions and the HCT-116 colon cell line. FIGS. 9Dand 9E demonstrate that fraction E was active at an IC₅₀ value ofapproximately 400 μg/mL. This value was obtained by extrapolation of theexisting curve. Note that the slope of the dose response curve forfraction D also indicated activity. However, no IC₅₀ value wasdetermined from this plot, since the slope of the curve was too shallowto obtain a reliable value. FIGS. 9F-9H depict representative resultsobtained from the fraction combination study. In this case, procyanidinfraction combination B+D did not show appreciable activity, whereasfraction combinations A+E and D+E were active at IC₅₀ values of 500μg/mL and 85 μg/mL, respectively. The IC₅₀ values that were obtainedfrom dose response curves of other fraction combinations averaged about250 μg/mL when fraction E was present. The extrapolated IC₅₀ values arelisted in Table 6.

[0342] D. ACHN Renal Cell Line

[0343] FIGS. 10A-10H show the typical dose response relationshipsbetween cocoa procyanidin fractions and the ACHN renal cell line. FIGS.10A-10E indicated that no individual fraction was active against thiscell line. FIGS. 10F-10H depict representative results obtained from thefraction combination study. In this case, procyanidin fractioncombination B+C was inactive, whereas the fraction combination A+Eresulted in an extrapolated IC₅₀ value of approximately 500 μg/mL. Doseresponse curves similar to the C+D combination were considered inactive,since their slopes were too shallow. Extrapolated IC₅₀ values for otherfraction combinations are listed in Table 6.

[0344] E. A-549 Lung Cell Line

[0345] FIGS. 11A-11H show the typical dose response relationshipsbetween cocoa procyanidin fractions and the A-549 lung cell line. Noactivity could be detected from any individual fraction or combinationof fractions at the doses used in the assay. However, procyanidinfractions may nonetheless have utility with respect to this cell line.

[0346] F. SK-5 Melanoma Cell Line

[0347] FIGS. 12A-12H show the typical dose response relationshipsbetween cocoa procyanidin fractions and the SK-5 melanoma cell line. Noactivity could be detected from any individual fraction or combinationof fractions at the doses used in the assay. However, procyanidinfractions may nonetheless have utility with respect to this cell line.

[0348] G. MCF-7 Breast Cell Line

[0349] FIGS. 13A-13H show the typical dose response relationshipsbetween cocoa procyanidin fractions and the MCF-7 breast cell line. Noactivity could be detected from any individual fraction or combinationof fractions at the doses used in the assay. However, procyanidinfractions may nonetheless have utility with respect to this cell line.

[0350] H. CCRF-CEM T-Cell Leukemia Line

[0351] A typical dose response curves were originally obtained againstthe CCRF-CEM T-cell leukemia line. However, microscopic counts of cellnumber versus time at different fraction concentrations indicated that500 μg of fractions A, B and D effected an 80% growth reduction over afour day period. A representative dose response relationship is shown inFIG. 14.

[0352] I. Summary

[0353] The IC₅₀ values obtained from these assays are collectivelylisted in Table 6 for all the cell lines except for CCRF-CEM T-cellleukemia. The T-cell leukemia data was intentionally omitted from theTable, since a different assay procedure was used. A general summary ofthese results indicated that the most activity was associated withfractions D and E. These fractions were most active against the PC-3(prostate) and KB (nasopharyngeal/HeLa) cell lines. These fractions alsoevidenced activity against the HCT-116 (colon) and ACHN (renal) celllines, albeit but only at much higher doses. No activity was detectedagainst the MCF-7 (breast), SK-5 (melanoma) and A-549 (lung) cell lines.However, procyanidin fractions may nonetheless have utility with respectto these cell lines. Activity was also shown against the CCRF-CEM(T-cell leukemia) cell line. It should also be noted that fractions Dand E are the most complex compositionally. Nonetheless, from this datait is clear that cocoa extracts, especially cocoa procyanidins, havesignificant anti-tumor, anti-cancer or antineoplastic activity. TABLE 6IC₅₀ Values for Cocoa Procyanidin Fractions Against Various Cell Lines(IC₅₀ values in μg/mL) A- FRACTION PC-3 KB HCT-116 ACHN MCF-7 SK-5 549 AB C D 90 80 E 75 75 400 A + B A + C 125 100 A + D 75 75 A + E 80 75 500500 B + C B + D 75 80 B + E 60 65 200 C + D 80 75 1000 C + E 80 70 250D + E 80 60 85

Example 8 Anti-Cancer, Anti-Tumor or Antineoplastic Activity of CocoaExtracts (Procyanidins)

[0354] Several additional in vitro assay procedures were used tocomplement and extend the results presented in Examples 6 and 7.

[0355] Method A. Crystal Violet Staining Assay

[0356] All human tumor cell lines were obtained from the American TypeCulture Collection. Cells were grown as monolayers in IMEM containing10% fetal bovine serum without antibiotics. The cells were maintained ina humidified, 5% CO₂ atmosphere at 37° C.

[0357] After trypsinization, the cells were counted and adjusted to aconcentration of 1,000-2,000 cells per 100 mL. Cell proliferation wasdetermined by plating the cells (1,000-2,000 cells/well) in a 96 wellmicrotiter plate. After addition of 100 μL cells per well, the cellswere allowed to attach for 24 hours. At the end of the 24 hour period,various cocoa fractions were added at different concentrations to obtaindose response results. The cocoa fractions were dissolved in media at a2 fold concentration and 100 μL of each solution was added in triplicatewells. On consecutive days, the plates were stained with 50 μL crystalviolet (2.5 g crystal violet dissolved in 125 mL methanol, 375 mLwater), for 15 min. The stain was removed and the plate was gentlyimmersed into cold water to remove excess stain. The washings wererepeated two more times, and the plates allowed to dry. The remainingstain was solubilized by adding 100 μL of 0.1M sodium citrate/50%ethanol to each well. After solubilization, the number of cells werequantitated on an ELISA plate reader at 540 nm (reference filter at 410nm). The results from the ELISA reader were graphed with absorbance onthe y-axis and days growth on the x-axis.

[0358] Method B. Soft Agar Cloning Assay

[0359] Cells were cloned in soft agar according to the method describedby Nawata et al. (1981). Single cell suspensions were made in mediacontaining 0.8% agar with various concentrations of cocoa fractions. Thesuspensions were aliquoted into 35 mm dishes coated with mediacontaining 1.0% agar. After 10 days incubation, the number of coloniesgreater than 60 μm in diameter were determined on an Ominicron 3600Image Analysis System. The results were plotted with number of colonieson the y-axis and the concentrations of a cocoa fraction on the x-axis.

[0360] Method C. XTT-Microculture Tetrazolium Assay

[0361] The XTT assay procedure described by Scudiero et al. (1988) wasused to screen various cocoa fractions. The XTT assay was essentiallythe same as that described using the MTT procedure (Example 6) exceptfor the following modifications. XTT((2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino)carbonyl)-2H-tetrazoliumhydroxide) was prepared at 1 mg/mL medium without serum, prewarmed to37° C. PMS was prepared at 5 mM PBS. XTT and PMS were mixed together; 10μL of PMS per mL XTT and 50 μL PMS-XTT were added to each well. After anincubation at 37° C. for 4 hr, the plates were mixed 30 min. on amechanical shaker and the absorbance measured at 450-600 nm. The resultswere plotted with the absorbance on the y-axis and days growth orconcentration on the x-axis.

[0362] For methods A and C, the results were also plotted as the percentcontrol as the y-axis and days growth or concentration on the x-axis.

[0363] A comparison of the XTT and Crystal Violet Assay procedures wasmade with cocoa fraction D & E (Example 3B) against the breast cancercell line MCF-7 p168 to determine which assay was most sensitive. Asshown in FIG. 15A, both assays showed the same dose-response effects forconcentrations >75 μg/mL. At concentrations below this value, thecrystal violet assay showed higher standard deviations than the XTTassay results. However, since the crystal violet assay was easier touse, all subsequent assays, unless otherwise specified, were performedby this procedure.

[0364] Crystal violet assay results are presented (FIGS. 15B-15E) todemonstrate the effect of a crude polyphenol extract (Example 2) on thebreast cancer cell line MDA MB231, prostate cancer cell line PC-3,breast cancer cell line MCF-7 p163, and cervical cancer cell line Hela,respectively. In all cases a dose of 250 μg/mL completely inhibited allcancer cell growth over a period of 5-7 days. The Hela cell lineappeared to be more sensitive to the extract, since a 100 μg/mL dosealso inhibited growth. Cocoa fractions from Example 3B were also assayedagainst Hela and another breast cancer cell line SKBR-3. The results(FIGS. 15F and 15G) showed that fraction D & E has the highest activity.As shown in FIGS. 15H and 15I, IC₅₀ values of about 40 μg/mL D & E wereobtained from both cancer cell lines.

[0365] The cocoa fraction D & E was also tested in the soft agar cloningassay which determines the ability of a test compound(s) to inhibitanchorage independent growth. As shown in FIG. 15J, a concentration of100 μg/mL completely inhibited colony formation of Hela cells.

[0366] Crude polyphenol extracts obtained from eight different cocoagenotypes representing the three horticultural races of cocoa were alsoassayed against the Hela cell line. As shown in FIG. 15K all cocoavarieties showed similar dose-response effects. The UIT-1 varietyexhibited the most activity against the Hela cell line. These resultsdemonstrated that all cocoa genotypes possess a polyphenol fraction thatelicits activity against at least one human cancer cell line that isindependent of geographical origin, horticultural race, and genotype.

[0367] Another series of assays were performed on crude polyphenolextracts prepared on a daily basis from a one ton scale traditional5-day fermentation of Brazilian cocoa beans, followed by a 4-day sundrying stage. The results shown in FIG. 15L showed no obvious effect ofthese early processing stages, suggesting little change in thecomposition of the polyphenols. However, it is known (Lehrian andPatterson, 1983) that polyphenol oxidase (PPO) will oxidize polyphenolsduring the fermentation stage. To determine what effect enzymaticallyoxidized polyphenols would have on activity, another experiment wasperformed. Crude PPO was prepared by extracting finely ground,unfermented, freeze dried, defatted Brazilian cocoa beans with acetoneat a ratio of 1 gm powder to 10 mL acetone. The slurry was centrifugedat 3,000 rpm for 15 min. This was repeated three times, discarding thesupernatant each time with the fourth extraction being poured through aBuchner filtering funnel. The acetone powder was allowed to air dry,followed by assay according to the procedures described by McLord andKilara, (1983). To a solution of crude polyphenols (100 mg/10 mLCitrate-Phosphate buffer, 0.02M, pH 5.5) 100 mg of acetone powder (4,000units activity/mg protein) was added and allowed to stir for 30 min.with a stream of air bubbled through the slurry. The sample wascentrifuged at 5,000×g for 15 min. and the supernatant extracted 3× with20 mL ethyl acetate. The ethyl acetate extracts were combined, taken todryness by distillation under partial vacuum and 5 mL water added,followed by lyophilization. The material was then assayed against Helacells and the dose-response compared to crude polyphenol extracts thatwere not enzymatically treated. The results (FIG. 15M) showed asignificant shift in the dose-response curve for the enzymaticallyoxidized extract, showing that the oxidized products were moreinhibitory than their native forms.

Example 9 Antioxidant Activity of Cocoa Extracts Containing Procyanidins

[0368] Evidence in the literature suggests a relationship between theconsumption of naturally occurring antioxidants (Vitamins C, E andBeta-carotene) and a lowered incidence of disease, including cancer(Designing Foods, 1993; Caragay, 1992). It is generally thought thatthese antioxidants affect certain oxidative and free radical processesinvolved with some types of tumor promotion. Additionally, some plantpolyphenolic compounds that have been shown to be anticarcinogenic, alsopossess substantial antioxidant activity (Ho et al., 1992; Huang et al.,1992).

[0369] To determine whether cocoa extracts containing procyanidinspossessed antioxidant properties, a standard Rancimat method wasemployed. The procedures described in Examples 1, 2 and 3 were used toprepare cocoa extracts which were manipulated further to produce twofractions from gel permeation chromatography. These two fractions areactually combined fractions A through C, and D and E (See FIG. 1) whoseantioxidant properties were compared against the synthetic antioxidantsBHA and BHT.

[0370] Peanut Oil was pressed from unroasted peanuts after the skinswere removed. Each test compound was spiked into the oil at two levels,˜100 ppm and ˜20 ppm, with the actual levels given in Table 7. 50 μL ofmethanol solubilized antioxidant was added to each sample to aid indispersion of the antioxidant. A control sample was prepared with 50 μLof methanol containing no antioxidant.

[0371] The samples were evaluated in duplicate, for oxidative stabilityusing the Rancimat stability test at 100° C. and 20 cc/min of air.Experimental parameters were chosen to match those used with the ActiveOxygen Method (AOM) or Swift Stability Test (Van Oosten et al., 1981). Atypical Rancimat trace is shown in FIG. 16. Results are reported inTable 8 as hours required to reach a peroxide level of 100 meq. TABLE 7Concentrations of Antioxidants LEVEL 1 LEVEL 2 SAMPLE ppm ButylatedHydroxytoluene 24 120 (BHT) Butylated Hydroxyanisole 24 120 (BHA) CrudeEthyl Acetate Fraction 22 110 of Cocoa Fraction A-C 20 100 Fraction D-E20 100

[0372] TABLE 8 Oxidative Stability of Peanut Oil with VariousAntioxidants 20 ppm 100 ppm SAMPLE average Control 10.5 ± 0.7 BHT 16.5 ±2.1 12.5 ± 2.1 BHA 13.5 ± 2.1 14.0 ± 1.4 Crude Cocoa Fraction 18.0 ± 0.019.0 ± 1.4 Fraction A-C 16.0 ± 6.4 17.5 ± 0.0 Fraction D-E 14.0 ± 1.412.5 ± 0.7

[0373] These results demonstrated increased oxidative stability ofpeanut oil with all of the additives tested. The highest increase inoxidative stability was realized by the sample spiked with the crudeethyl acetate extract of cocoa. These results demonstrated that cocoaextracts containing procyanidins have antioxidant potential equal to orgreater than equal amounts of synthetic BHA and BHT. Accordingly, theinvention may be employed in place of BHT or BHA in known utilities ofBHA or BHT, for instance as an antioxidant and/or food additive. And, inthis regard, it is noted too that the invention is from an ediblesource. Given these results, the skilled artisan can also readilydetermine a suitable amount of the invention to employ in such “BHA orBHT” utilities, e.g., the quantity to add to food, without undueexperimentation.

Example 10 Topoisomerase II Inhibition Study

[0374] DNA topoisomerase I and II are enzymes that catalyze the breakingand rejoining of DNA strands, thereby controlling the topological statesof DNA (Wang, 1985). In addition to the study of the intracellularfunction of topoisomerase, one of the most significant findings has beenthe identification of topoisomerase II as the primary cellular targetfor a number of clinically important antitumor compounds (Yamashita etal., 1990) which include intercalating agents (m-AMSA, Adriamycin® andellipticine) as well as nonintercalating epipodophyllotoxins. Severallines of evidence indicate that some antitumor drugs have the commonproperty of stabilizing the DNA-topoisomerase II complex (“cleavablecomplex”) which upon exposure to denaturing agents results in theinduction of DNA cleavage (Muller et al., 1989). It has been suggestedthat the cleavable complex formation by antitumor drugs produces bulkyDNA adducts that can lead to cell death.

[0375] According to this attractive model, a specific new inducer of DNAtopoisomerase II cleavable complex is useful as an anti-cancer,anti-tumor or antineoplastic agent. In an attempt to identify cytotoxiccompounds with activities that target DNA, the cocoa procyanidins werescreened for enhanced cytotoxic activity against several DNA-damagesensitive cell lines and enzyme assay with human topoisomerase IIobtained from lymphoma.

[0376] A. Decatenation of Kinetoplast DNA by Topoisomerase II

[0377] The in vitro inhibition of topoisomerase II decatenation ofkinetoplast DNA, as described by Muller et al. (1989), was performed asfollows. Nuclear extracts containing topoisomerase II activity wereprepared from human lymphoma by modifications of the methods of Milleret al. (1981) and Danks et al. (1988). One unit of purified enzyme wasenough to decatenate 0.25 μg of kinetoplast DNA in 30 min. at 34° C.Kinetoplast DNA was obtained from the trypanosome Crithidia fasciculata.Each reaction was carried out in a 0.5 mL microcentrifuge tubecontaining 19.5 μL H₂O, 2.5 μL 10×buffer (1×buffer contains 50 mMtris-HCl, pH 8.0, 120 mM KCl, 10 mM MgCl₂, 0.5 mM ATP, 0.5 mMdithiothreitol and 30 μg BSA/mL), 1 μL kinetoplast DNA (0.2 μg), and 1μL DMSO-containing cocoa procyanidin test fractions at variousconcentrations. This combination was mixed thoroughly and kept on ice.One unit of topoisomerase was added immediately before incubation in awaterbath at 34° C. for 30 min.

[0378] Following incubation, the decatenation assay was stopped by theaddition of 5 μL stop buffer (5% sarkosyl, 0.0025% bromophenol blue, 25%glycerol) and placed on ice. DNA was electrophoresed on a 1% agarose gelin TAE buffer containing ethidium bromide (0.5 μg/mL). Ultravioletillumination at 310 nm wavelength allowed the visualization of DNA. Thegels were photographed using a Polaroid Land camera.

[0379]FIG. 17 shows the results of these experiments. Fully catenatedkinetoplast DNA does not migrate into a 1% agarose gel. Decatenation ofkinetoplast DNA by topoisomerase II generates bands of monomeric DNA(monomer circle, forms I and II) which do migrate into the gel.Inhibition of the enzyme by addition of cocoa procyanidins is apparentby the progressive disappearance of the monomer bands as a function ofincreasing concentration. Based on these results, cocoa procyanidinfractions A, B, D, and E were shown to inhibit topoisomerase II atconcentrations ranging from 0.5 to 5.0 μg/mL. These inhibitorconcentrations were very similar to those obtained for mitoxanthrone andm-AMSA (4′-(9-acridinylamino)methanesulfon-m-anisidide).

[0380] B. Drug Sensitive Cell Lines

[0381] Cocoa procyanidins were screened for cytotoxicity against severalDNA-damage sensitive cell lines. One of the cell lines was the xrs-6 DNAdouble strand break repair mutant developed by P. Jeggo (Kemp et al.,1984). The DNA repair deficiency of the xrs-6 cell line renders themparticularly sensitive to x-irradiation, to compounds that produce DNAdouble strand breaks directly, such as bleomycin, and to compounds thatinhibit topoisomerase II, and thus may indirectly induce double strandbreaks as suggested by Warters et al. (1991). The cytotoxicity towardthe repair deficient line was compared to the cytotoxicity against a DNArepair proficient CHO line, BR1. Enhanced cytotoxicity towards therepair deficient (xrs-6) line was interpreted as evidence for DNAcleavable double strand break formation.

[0382] The DNA repair competent CHO line, BR1, was developed by Barrowset al. (1987) and expresses 0⁶-alkylguanine-DNA-alkyltransferase inaddition to normal CHO DNA repair enzymes. The CHO double strand breakrepair deficient line (xrs-6) was a generous gift from Dr. P. Jeggo andco-workers (Jeggo et al., 1989). Both of these lines were grown asmonolayers in alpha-MEM containing serum and antibiotics as described inExample 6. Cells were maintained at 37° C. in a humidified 5% CO₂atmosphere. Before treatment with cocoa procyanidins, cells grown asmonolayers were detached with trypsin treatment. Assays were performedusing the MTT assay procedure described in Example 6.

[0383] The results (FIG. 18) indicated no enhanced cytotoxicity towardsthe xrs-6 cells suggesting that the cocoa procyanidins inhibitedtopoisomerase II in a manner different from cleavable double strandbreak formation. That is, the cocoa procyanidins interact withtopoisomerase II before it has interacted with the DNA to form anoncleavable complex.

[0384] Noncleavable complex forming compounds are relatively newdiscoveries. Members of the anthracyclines, podophyllin alkaloids,anthracenediones, acridines, and ellipticines are all approved forclinical anti-cancer, anti-tumor or antineoplastic use, and they producecleavable complexes (Liu, 1989). Several new classes of topoisomerase IIinhibitors have recently been identified which do not appear to producecleavable complexes. These include amonafide (Hsiang et al., 1989),distamycin (Fesen et al., 1989), flavanoids (Yamashita et al., 1990),saintopin (Yamashita et al., 1991), membranone (Drake et al., 1989),terpenoids (Kawada et al., 1991), anthrapyrazoles (Fry et al., 1985),dioxopiperazines (Tanabe et al., 1991), and the marine acridine-dercitin(Burres et al., 1989).

[0385] Since the cocoa procyanidins inactivate topoisomerase II beforecleavable complexes are formed, they have chemotherapy value eitheralone or in combination with other known and mechanistically definedtopoisomerase II inhibitors. Additionally, cocoa procyanidins alsoappear to be a novel class of topoisomerase II inhibitors, (Kashiwada etal., 1993) and may thus be less toxic to cells than other knowninhibitors, thereby enhancing their utility in chemotherapy.

[0386] The human breast cancer cell line MCF-7 (ADR) which expresses amembrane bound glycoprotein (gp170) to confer multi-drug resistance(Leonessa et al., 1994) and its parental line MCF-7 p168 were used toassay the effects of cocoa fraction D & E. As shown in FIG. 19, theparental line was inhibited at increasing dose levels of fraction D & E,whereas the Adriamycin (ADR) resistant line was less effected at thehigher doses. These results show that cocoa fraction D & E has an effecton multi-drug resistant cell lines.

Example 11 Synthesis of Procyanidins

[0387] The synthesis of procyanidins was performed according to theprocedures developed by Delcour et al. (1983), with modification. Inaddition to condensing (+)-catechin with dihydroquercetin under reducingconditions, (−)-epicatechin was also used to reflect the highconcentrations of (−)-epicatechin that naturally occur in unfermentedcocoa beans. The synthesis products were isolated, purified, analyzed,and identified by the procedures described in Examples 3, 4 and 5. Inthis manner, the biflavanoids, triflavanoids and tetraflavanoids areprepared and used as analytical standards and, in the manner describedabove with respect to cocoa extracts.

Example 12 Assay of Normal Phase Semi-Preparative Fractions

[0388] Since the polyphenol extracts are compositionally complex, it wasnecessary to determine which components were active against cancer celllines for further purification, dose-response assays and comprehensivestructural identification. A normal phase semi preparative HPLCseparation (Example 3B) was used to separate cocoa procyanidins on thebasis of oligomeric size. In addition to the original extract, twelvefractions were prepared (FIGS. 2B and 15 O) and assayed at 100 μg/mL and25 μg/mL doses against Hela and SKBR-3 cancer cell lines to determinewhich oligomer possessed the greatest activity. As shown in FIGS. 20Aand B, fractions 4-11 (pentamer-dodecamer) significantly inhibited HeLaand SKBr-3 cancer cell lines at the 100 μg/mL level. These resultsindicated that these specific oligomers had the greatest activityagainst Hela and SKBR-3 cells. Additionally, normal phase HPLC analysisof cocoa fraction D & E indicated that this fraction, used in previousinvestigations, e.g., Example 7, was enriched with these oligomers.

Example 13 HPLC Purification Methods

[0389] Method A. GPC Purification

[0390] Procyanidins obtained as in Example 2 were partially purified byliquid chromatography on Sephadex LH 20 (72.5×2.5 cm), using 100%methanol as the eluting solvent, at a flow rate of 3.5 mL/min. Fractionsof the eluent were collected after the first 1.5 hours, and thefractions were concentrated by a rotary evaporator, redissolved in waterand freeze dried. These fractions were referred to as pentamer enrichedfractions. Approximately 2.00 g of the extract obtained from Example 2was subfractionated in this manner. Results are shown in Table 9. TABLE9 Composition of Fractions Obtained: Mono- Hexa- Fraction mer (% DimerTrimer Tetramer Pentamer mer (% Heptamer Octamer Nonamer DecamerUndecamer Others (Time) Area) (% Area) (% Area) (% Area) (% Area) Area)(% Area) (% Area) (% Area) (% Area) (% Area) (% Area) 1:15 73 8 16 3 NDND ND ND ND ND ND ND 1:44 67 19 10 3 1 tr tr tr tr tr tr tr 2:13 30 2924 11 4 1 tr tr tr tr tr tr 2:42 2 16 31 28 15 6 2 tr tr tr tr tr 3:11 112 17 25 22 13 7 2 1 tr tr tr 3:40 tr 18 13 18 20 15 10 5 2 tr tr tr4:09 tr 6 8 17 21 19 14 8 4 2 tr tr

[0391] Method B. Normal Phase Separation

[0392] Procyanidins obtained as Example 2 were separated purified bynormal phase chromatography on Supelcosil LC-Si, 100 Å, 5 μm (250×4.6mm), at a flow rate of 1.0 mL/min, or, in the alternative, Lichrosphere®Silica 100, 100 Å, 5 μm (235×3.2 mm), at a flow rate of 0.5 mL/min.Separations were aided by a step gradient under the followingconditions: (Time, %A, %B); (0, 82, 14), (30, 67.6, 28.4), (60, 46, 50),(65, 10, 86), (70, 10, 86). Mobile phase composition wasA=dichloromethane; B=methanol; and C=acetic acid:water (1:1). Componentswere detected by fluorescence where λ_(ex)=276 nm and λ_(em)=316 nm, andby UV at 280 nm. The injection volume was 5.0 μL (20 mg/mL) of theprocyanidins obtained from Example 2. These results are shown in FIGS.40A and 40B.

[0393] In the alternative, separations were aided by a step gradientunder the following conditions: (Time, %A, %B); (0, 76, 20); (25, 46,50); (30, 10, 86). Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). The results are shown inFIGS. 41A and 41B.

[0394] Method C. Reverse-Phase Separation

[0395] Procyanidins obtained as in Example 2 were separated purified byreverse phase chromatography on Hewlett Packard Hypersil ODS 5 μm.(200×2.1 mm), and a Hewlett Packard Hypersil ODS 5 μm guard column(20×2.1 mm). The procyanidins were eluted with a linear gradient of 20%B into A in 20 minutes, followed by a column wash with 100% B at a flowrate of 0.3 mL/min. The mobile phase composition was a degassed mixtureof B=1.0% acetic acid in methanol and A=2.0% acetic acid in nanopurewater. Components were detected by UV at 280 nm, and fluorescence whereλ_(ex)=276 nm and λ_(em)=316 nm; and the injection volume was 2.0 μL (20mg/mL).

Example 14 HPLC Separation of Pentamer Enriched Fractions

[0396] Method A. Semi-Preparative Normal Phase HPLC

[0397] The pentamer enriched fractions were further purified bysemi-preparative normal phase HPLC by a Hewlett Packard 1050 HPLC systemequipped with a Millipore-Waters model 480 LC detector set at 254 nm,which was assembled with a Pharmacia Frac-100 Fraction Collector set topeak mode. Separations were effected on a Supelco 5 μm Supelcosel LC-Si,100 Å column (250×10 mm) connected with a Supelco 5μ Supelguard LC-Siguard column (20×4.6 mm). Procyanidins were eluted by a linear gradientunder the following conditions: (Time, %A, %B); (0, 82, 14), (30, 67.6,28.4), (60, 46, 50), (65, 10, 86), (70, 10, 86) followed by a 10 minutere-equilibration. Mobile phase composition was A=dichloromethane;B=methanol; and C=acetic acid:water (1:1). A flow rate of 3 mL/min wasused. Components were detected by UV at 254 nm; and recorded on a Kipp &Zonan BD41 recorder. Injection volumes ranged from 100-250 μl of 10 mgof procyanidin extracts dissolved in 0.25 mL 70% aqueous acetone.Individual peaks or select chromatographic regions were collected ontimed intervals or manually by fraction collection for furtherpurification and subsequent evaluation. HPLC conditions: 250 × 100 mmSupelco Supelcosil LC-Si (5 μm) Semipreparative Column 20 × 4.6 mmSupelco Supelcosil LC-Si (5 μm) Guard Column Detector: Waters LCSpectrophotometer Model 480 @ 254 nm Flow rate: 3 mL/min., ColumnTemperature: ambient, Injection: 250 μL of pentamer enriched extractacetic acid: Gradient: CH₂Cl₂ methanol water (1:1) 0 82 14 4 30 67.628.4 4 60 46 50 4 65 10 86 4 70 10 86 4

[0398] Method B. Reverse Phase Separation

[0399] Procyanidin extracts obtained as in Example 13 were filteredthrough a 0.45μ nylon filter and analyzed by a Hewlett Packard 1090ternary phase HPLC system equipped with a Diode Array detector and a HPmodel 1046A Programmable Fluorescence Detector. Separations wereeffected at 45° C. on a Hewlett Packard 5μ Hypersil ODS column (200×2.1mm). The procyanidins were eluted with a linear gradient of 60% B into Afollowed by a column wash with B at a flow rate of 0.3 mL/min. Themobile phase composition was a de-gassed mixture of B=0.5% acetic acidin methanol and A=0.5% acetic acid in nanopure water. Acetic acid levelsin A and B mobile phases can be increased to 2%. Components weredetected by fluorescence, where λ_(ex)=276 nm and λ_(em)=316 nm, and byUV at 280 nm. Concentrations of (+)-catechin and (−)-epicatechin weredetermined relative to reference standard solutions. Procyanidin levelswere estimated by using the response factor for (−)-epicatechin.

[0400] Method C. Normal Phase Separation

[0401] Pentamer enriched procyanidin extracts obtained as in Example 13were filtered through a 0.45μ nylon filter and analyzed by a HewlettPackard 1090 Series II HPLC system equipped with a HP Model 1046AProgrammable Fluorescence detector and Diode Array detector. Separationswere effected at 37° C. on a 5μ Phenomenex Lichrosphere® Silica 100column (250×3.2 mm) connected to a Supelco Supelguard LC-Si 5μ guardcolumn (20×4.6 mm). Procyanidins were eluted by linear gradient underthe following conditions: (time, %A, %B); (0, 82, 14), (30, 67.6, 28.4),(60, 46, 50), (65, 10, 86), (70, 10, 86), followed by an 8 minutere-equilibration. Mobile phase composition was A=dichloromethane,B=methanol, and C=acetic acid:water at a volume ratio of 1:1. A flowrate of 0.5 mL/min was used. Components were detected by fluorescence,where λ_(ex)=276 nm and λ_(em)=316 nm or by UV at 280 nm. Arepresentative HPLC chromatogram showing the separation of the variousprocyanidins is shown in FIG. 2 for one genotype. Similar HPLC profileswere obtained from other Theobroma, Herrania and/or their inter or intraspecific crosses. HPLC conditions: 250 × 3.2 mm PhenomenexLichrosphere ™ Silica 100 column (5μ) 20 × 4.6 mm Supelco SupelguardLC-Si (5 μm) guard column Detector: Photodiode Array @ 280 nmFluorescence λ_(ex) = 276 nm; λ_(em) = 316 nm Flow rate: 0.5 mL/min.,Column Temperature: 37° C. acetic acid: Gradient: CH₂Cl₂ methanol water(1:1) 0 82 14 4 30 67.6 28.4 4 60 46 50 4 65 10 86 4 70 10 86 4

[0402] Method D. Preparative Normal Phase Separation

[0403] The pentamer enriched fractions obtained as in Example 13 werefurther purified by preparative normal phase chromatography by modifyingthe method of Rigaud et al., (1993) J. Chrom. 654, 255-260.

[0404] Separations were affected at ambient temperature on a 5μSupelcosil LC-Si 100 Å column (50×2 cm), with an appropriate guardcolumn. Procyanidins were eluted by a linear gradient under thefollowing conditions: (time, %A, %B, flow rate); (0, 92.5, 7.5, 10);(10, 92.5, 7.5, 40); (30, 91.5, 18.5, 40); (145, 88, 22, 40); (150, 24,86, 40); (155, 24, 86, 50); (180, 0, 100, 50). Prior to use, the mobilephase components were mixed by the following protocol:

[0405] Solvent A preparation (82% CH₂Cl₂, 14% methanol, 2% acetic acid,2% water):

[0406] 1. Measure 80 mL of water and dispense into a 4 L bottle.

[0407] 2. Measure 80 mL of acetic acid and dispense into the same 4 Lbottle.

[0408] 3. Measure 560 mL of methanol and dispense into the same 4 Lbottle.

[0409] 4. Measure 3280 mL of methylene chloride and dispense into the 4L bottle.

[0410] 5. Cap the bottle and mix well.

[0411] 6. Purge the mixture with high purity Helium for 5-10 minutes todegas.

[0412] Repeat steps 1-6 two times to yield 8 volumes of solvent A.

[0413] Solvent B preparation (96% methanol, 2% acetic acid, 2% water):

[0414] 1. Measure 80 mL of water and dispense into a 4 L bottle.

[0415] 2. Measure 80 mL of acetic acid and dispense into the same 4 Lbottle.

[0416] 3. Measure 3840 mL of methanol and dispense 3840 mL of methanoland dispense into the same 4 L bottle.

[0417] 4. Cap the bottle and mix well.

[0418] 5. Purge the mixture with high purity Helium for 5-10 minutes todegas.

[0419] Repeat steps 1-5 to yield 4 volumes of solvent B. Mobile phasecomposition was A=methylene chloride with 2% acetic acid and 2% water;B=methanol with 2% acetic acid and 2% water. The column load was 0.7 gin 7 mL. components were detected by UV at 254 nm. A typical preparativenormal phase HPLC separation of cocoa procyanidins is shown in FIG. 42.HPLC Conditions: Column: 50 × 2 cm 5μ Supelcosil LC-Si run @ ambienttemperature. Mobile Phase: A = Methylene Chloride with 2% Acetic Acidand 2% Water. B = Methanol with 2% Acetic Acid and 2% Water.Gradient/Flow Profile: TIME FLOW RATE (MIN) % A % B (mL/min) 0 92.5 7.510 10 92.5 7.5 40 30 91.5 8.5 40 145 88.0 22.0 40 150 24.0 86.0 40 15524.0 86.0 50 180 0.0 100.0 50

Example 15 Identification of Procyanidins

[0420] Procyanidins obtained as in Example 14, method D were analyzed byMatrix Assisted Laser Desorption Ionization-Time of Flight/MassSpectrometry (MALDI-TOF/MS) using a HP G2025A MALDI-TOF/MS systemequipped with a Lecroy 9350 500 MHz Oscilloscope. The instrument wascalibrated in accordance with the manufacturer's instructions with a lowmolecular weight peptide standard (HP Part No. G2051A) or peptidestandard (HP Part No. G2052A) with 2,5-dihydroxybenzoic acid (DHB) (HPPart No. G2056A) as the sample matrix. One (1.0) mg of sample wasdissolved in 500 μL of 70/30 methanol/water, and the sample was thenmixed with DHB matrix, at a ratio of 1:1, 1:10 or 1:50 (sample:matrix)and dried on a mesa under vacuum. The samples were analyzed in thepositive ion mode with the detector voltage set at 4.75 kV and the laserpower set between 1.5 and 8 μJ. Data was collected as the sum of anumber of single shots and displayed as units of molecular weight andtime of flight. A representative MALDI-TOF/MS is shown in FIG. 22A.

[0421] FIGS. 22 and C show MALDI-TOF/MS spectra obtained from partiallypurified procyanidins prepared as described in Example 3, Method A andused for in vitro assessment as described in Examples 6 and 7, and whoseresults are summarized in Table 6. This data illustrates that theinventive compounds described herein were predominantly found infractions D-E, but not A-C.

[0422] The spectra were obtained as follows:

[0423] The purified D-E fraction was subjected to MALDI-TOF/MS asdescribed above, with the exception that the fraction was initiallypurified by SEP-PACK® C-18 cartridge. Five (5) mg of fraction D-E in 1mL nanopure water was loaded onto a pre-equilibrated SEP-PACK®cartridge. The column was washed with 5 mL nanopure water to eliminatecontaminants, and procyanidins were eluted with 1 mL 20% methanol.Fractions A-C were used directly, as they were isolated in Example 3,Method A, without further purification.

[0424] These results confirmed and extended earlier results (see Example5, Table 3, FIGS. 20A and B) and indicate that the inventive compoundshave utility as sequestrants of cations. In particular, MALDI-TOF/MSresults conclusively indicated that procyanidin oligomers of n=5 andhigher (see FIGS. 20A and B; and formula under Objects and Summary ofthe Invention) were strongly associated with anti-cancer activity withthe HeLa and SKBR-3 cancer cell line model. Oligomers of n=4 or lesswere ineffective with these models. The pentamer structure apparentlyhas a structural motif which is present in it and in higher oligomerswhich provides the activity. Additionally, it was observed that theMALDI-TOF/MS data showed strong M⁺ ions of Na⁺, 2 Na⁺, K⁺, 2 K⁺, Ca⁺⁺,demonstrating the utility as cation sequestrants.

Example 16 Purification of Oligomeric Fractions

[0425] Method A. Purification by Semi-Preparative Reverse Phase HPLC

[0426] Procyanidins obtained from Example 14, Method A and B and D werefurther separated to obtain experimental quantities of like oligomersfor further structural identification and elucidation (e.g., Example 15,18, 19, and 20). A Hewlett Packard 1050 HPLC system equipped with avariable wavelength detector, Rheodyne 7010 injection valve with 1 mLinjection loop was assembled with a Pharmacia FRAC-100 FractionCollector. Separations were effected on a Phenomenex Ultracarb® 10μ ODScolumn (250×22.5 mm) connected with a Phenomenex 10μ ODS Ultracarb®(60×10 mm) guard column. The mobile phase composition was A=water;B=methanol used under the following linear gradient conditions: (time,%A); (0, 85), (60, 50), (90, 0 and (110, 0) at a flow rate of 5 mL/min.Individual peaks or select chromatographic regions were collected ontimed intervals or manually by fraction collection for furtherevaluation by MALDI-TOF/MS and NMR. Injection loads ranged from 25-100mg of material. A representative elution profile is shown in FIG. 23b.

[0427] Method B. Modified Semi-Preparative HPLC

[0428] Procyanidins obtained from Example 14, Method A and B and D werefurther separated to obtain experimental quantities of like oligomersfor further structural identification and elucidation (e.g., Example 15,18, 19, and 20). Supelcosil LC-Si 5μ column (250×10 mm) with aSupelcosil LC-Si 5μ (20×2 mm) guard column. The separations wereeffected at a flow rate of 3.0 mL/min, at ambient temperature. Themobile phase composition was A=dichloromethane; B=methanol; and C=aceticacid:water (1:1); used under the following linear gradient conditions:(time, %A, %B); (0, 82, 14); (22, 74, 21); (32, 74, 21); (60, 74, 50,4); (61, 82, 14), followed by column re-equilibration for 7 minutes.Injection volumes were 60 μL containing 12 mg of enriched pentamer.Components were detected by UV at 280 nm. A representative elutionprofile is shown in FIG. 23A.

Example 17 Molecular Modeling of Pentamers

[0429] Energy minimized structures were determined by molecular modelingusing Desktop Molecular Modeller, version 3.0, Oxford University Press,1994. Four representative views of [EC(4→8)]₄-EC (EC=epicatechin)pentamers based on the structure of epicatechin are shown in FIGS. 24A-D. A helical structure is suggested. In general when epicatechin isthe first monomer and the bonding is 4→8, a beta configuration results,when the first monomer is catechin and the bonding is 4→8, an alphaconfiguration results; and, these results are obtained regardless ofwhether the second monomer is epicatechin or catechin (an exception isent-EC(4→8)ent-EC). FIGS. 38A-38P show preferred pentamers, and, FIGS.39A to 39P show a library of stereoisomers up to and including thepentamer, from which other compounds within the scope of the inventioncan be prepared, without undue experimentation.

Example 18 NMR Evaluation of Pyrocyanidins

[0430]¹³C NMR spectroscopy was deemed a generally useful technique forthe study of procyanidins, especially as the phenols usually providegood quality spectra, whereas proton NMR spectra are considerablybroadened. The ¹³C NMR spectra of oligomers yielded useful informationfor A or B ring substitution patterns, the relative stereochemistry ofthe C ring and in certain cases, the position of the interflavanoidlinkages. Nonetheless, ¹H NMR spectra yielded useful information.

[0431] Further, HOHAHA, makes use of the pulse technique to transfermagnetization of a first hydrogen to a second in a sequence to obtaincross peaks corresponding to alpha, beta, gamma or delta protons. COSYis a 2D-Fourier transform NMR technique wherein vertical and horizontalaxes provide ¹H chemical shift and 1D spectra; and a point ofintersection provides a correlation between protons, whereby spin-spincouplings can be determined. HMQC spectra enhances the sensitivity ofNMR spectra of nuclei; other than protons and can reveal cross peaksfrom secondary and tertiary carbons to the respective protons. APT is a¹³C technique used in determining the number of hydrogens present at acarbon. An even number of protons at a carbon will result in a positivesignal, while an odd number of protons at a carbon will result in anegative signal.

[0432] Thus ¹³C NMR, ¹H NMR, HOHAHA (homonuclear Hartmann-Hahn), HMQC(heteronuclear multiple quantum coherence), COSY (Homonuclearcorrelation spectroscopy), APT (attached proton test), and XHCORR (avariation on HMQC) spectroscopy were used to elucidate the structures ofthe inventive compounds.

[0433] Method A. Monomer

[0434] All spectra were taken in deuterated methanol, at roomtemperature, at an approximate sample concentration of 10 mg/mL. Spectrawere taken on a Bruker 500 MHZ NMR, using methanol as an internalstandard.

[0435] FIGS. 44A-E represent the NMR spectra which were used tocharacterize the structure of the epicatechin monomer. FIG. 44A showsthe ¹H and ¹³C chemical shifts, in tabular form. FIGS. 44 B-E show ¹H,APT, XHCORR and COSY spectra for epicatechin.

[0436] Similarly, FIGS. 45A-F represent the NMR spectra which were usedto characterize the structure of the catechin monomer. FIG. 45A showsthe ¹H and ¹³C chemical shifts, in tabular form. FIGS. 44 B-F show ¹H,¹³C, APT, XHCORR and COSY spectra for catechin.

[0437] Method B. Dimers

[0438] All spectra were taken in 75% deuterated acetone in D₂O, usingacetone as an internal standard, and an approximate sample concentrationof 10 mg/mL.

[0439] FIGS. 46A-G represent the spectra which were used to characterizethe structure of the B2 dimer. FIG. 46A shows ¹H and ¹³C chemicalshifts, in tabular form. The terms T and B indicate the top half of thedimer and the bottom half of the dimer.

[0440]FIGS. 46B and C show the ¹³C and APT spectra, respectively, takenon a Bruker 500 MHZ NMR, at room temperature.

[0441] FIGS. 46D-G show the ¹H, HMQC, COSY and HOHAHA, respectively,which were taken on AMZ-360 MHZ NMR at a −7° C. The COSY spectrum wastaken using a gradient pulse.

[0442] FIGS. 47A-G represent the spectra which were used to characterizethe structure of the B5 dimer. FIG. 47A shows the ¹³C and ¹H chemicalshifts, in tabular form.

[0443] FIGS. 47B-D show the ¹H, ¹³C and APT, respectively, which weretaken on a Bruker 500 MHZ NMR, at room temperature.

[0444]FIG. 47E shows the COSY spectrum, taken on an AMX-360, at roomtemperature, using a gradient pulse.

[0445]FIGS. 47F and G show the HMQC and HOHAHA, respectively, taken onan AMX-360 MHZ NMR, at room temperature.

[0446] Method C. Trimer-Epicatechin/Catechin

[0447] All spectra were taken in 75% deuterated acetone in D₂O, at −3°C. using acetone as an internal standard, on an AMX-360 MHZ NMR, and anappropriate sample concentration of 10 mg/mL.

[0448] FIGS. 48A-D represent the spectra which were used to characterizethe structure of the epicatechin/catechin trimer. These figures show ¹H,COSY, HMQC and HOHAHA, respectively. The COSY spectrum was taken using agradient pulse.

[0449] Method D. Trimer-All Epicatechin

[0450] All spectra were taken in 70% deuterated acetone in D₂O, at −1.8°C., using acetone as an internal standard, on an AMX-360 MHZ NMR, and anappropriate sample concentration of 10 mg/mL.

[0451] FIGS. 49A-D represent the spectra which were used to characterizethe structure of all epicatechin trimer. These figures show ¹H, COSY,HMQC and HOHAHA, respectively. The COSY spectrum was taken using agradient pulse.

Example 19 Thiolysis of Procyanidins

[0452] In an effort to characterize the structure of procyanidins,benzyl mercaptan (BM) was reacted with catechin, epicatechin or dimersB2 and B5. Benzyl mercaptan, as well as phloroglucinol and thiophenol,can be utilized in the hydrolysis (thiolysis) of procyanidins in analcohol/acetic acid environment. Catechin, epicatechin or dimer (1:1mixture of B2 and B5 dimers) (2.5 mg) was dissolved in 1.5 mL ethanol,100 μL BM and 50 μL acetic acid, and the vessel (Beckman amino acidanalysis vessel) was evacuated and purged with nitrogen repeatedly untila final purge with nitrogen was followed by sealing the reaction vessel.The reaction vessel was placed in a heat block at 95° C., and aliquotsof the reaction were taken at 30, 60, 120 and 240 minutes. The relativefluorescence of each aliquot is shown in FIGS. 25A-C, representingepicatechin, catechin and dimers, respectively. Higher oligomers aresimilarly thiolyzed.

Example 20 Thiolysis and Desulfurization of Dimers

[0453] Dimers B2 and B5 were hydrolyzed with benzylmercaptan bydissolving dimer (B2 or B5; 1.0 mg) in 600 μl ethanol, 40 μL BM and 20μL acetic acid. The mixture was heated at 95° C. for 4 hours undernitrogen in a Beckman Amino Acid Analysis vessel. Aliquots were removedfor analysis by reverse-phase HPLC, and 75 μL of each of ethanol RaneyNickel and gallic acid (10 mg/mL) were added to the remaining reactionmedium in a 2 mL hypovial. The vessel was purged under hydrogen, andoccasionally shaken for 1 hour. The product was filtered through a 0.45μfilter and analyzed by reverse-phase HPLC. Representative elutionprofiles are shown in FIGS. 26 A and B. Higher oligomers are similarlydesulfurized. This data suggests polymerization of epicatechan orcatechin and therefore represents a synthetic route for preparation ofinventive compounds.

Example 21 In Vivo Activity of Pentamer in MDA MB 231 Nude Mouse Model

[0454] MDA-MB-231/LCC6 cell line. The cell line was grown in improvedminimal essential medium (IMEM) containing 10% fetal bovine serum andmaintained in a humidified, 5% CO₂ atmosphere at 37° C.

[0455] Mice. Female six to eight week old NCr nu/nu (athymic) mice werepurchased through NCI and housed in an animal facility and maintainedaccording to the regulations set forth by the United States Departmentof Agriculture, and the American Association for the Accreditation ofLaboratory Animal Care. Mice with tumors were weighed every other day,as well as weekly to determine appropriate drug dosing.

[0456] Tumor implantation. MDA-MD-231 prepared by tissue culture wasdiluted with IMEM to 3.3×10⁶ cells/mL and 0.15 mL (i.e. 0.5×10⁶ cells)were injected subcutaneously between nipples 2 and 3 on each side of themouse. Tumor volume was calculated by multiplying:length×width×height×0.5. Tumor volumes over a treatment group wereaveraged and Student's t test was used to calculate p values.

[0457] Sample preparation. Plasma samples were obtained by cardiacpuncture and stored at −70° C. with 15-20 mM EDTA for the purposes ofblood chemistry determinations. No differences were noted between thecontrol group and experimental groups.

[0458] Fifteen nude mice previously infected with 500,000 cellssubcutaneously with tumor cell line MDA-MB-231, were randomLy separatedinto three groups of 5 animals each and treated by intraperitonealinjection with one of: (i) placebo containing vehicle alone (DMSO); (ii)2 mg/mouse of purified pentameric procyanidin extract as isolated inExample 14 method D in vehicle (DMSO); and (iii) 10 mg/mouse purifiedpentameric procyanidin extract as isolated in Example 14, method D invehicle (DMSO).

[0459] The group (iii) mice died within approximately 48 to 72 hoursafter administration of the 10 mg, whereas the group (ii) mice appearednormal. The cause of death of the group (iii) mice was undetermined;and, cannot necessarily be attributed to the administration of inventivecompounds. Nonetheless, 10 mg was considered an upper limit with respectto toxicity.

[0460] Treatment of groups (i) and (ii) was repeated once a week, andtumor growth was monitored for each experimental and control group.After two weeks of treatment, no signs of toxicity were observed in themice of group (ii) and, the dose administered to this group wasincrementally increased by ½ log scale each subsequent week. Thefollowing Table represents the dosages administered during the treatmentschedule for mice of group (ii): Dose Week (mg/mouse) 1 2 2 2 3 4 4 5 55 6 5 7 5

[0461] The results of treatment are shown in FIGS. 27A and B and Table10. TABLE 10 IN VIVO ANTI-CANCER RESULTS % SURVIVAL % SURVIVAL %SURVIVAL DAY GROUP (i) GROUP (ii) GROUP (iii) 1 100 100 100 2 100 100100 3 100 100 0 4 100 100 5 100 100 6 100 100 7 100 100 8 100 100 9 100100 10 100 100 11 100 100 12 100 100 13 100 100 14 100 100 15 100 100 16100 100 17 100 100 18 100 100 19 100 100 20 100 100 21 100 100 22 75 10023 75 100 24 75 100 25 75 100 26 75 100 27 75 100 28 75 100 29 50 100 3050 100 31 50 100 32 50 100 33 50 100 34 50 100 35 50 100 36 25 100 37 25100 38 25 100 39 25 100 40 25 100 41 25 100 42 25 100 43 25 80 44 25 8045 25 80 46 25 80 47 25 80 48 25 80 49 25 80 50 25 60 51 25 60 52 25 6053 25 60 54 25 60 55 25 60 56 25 60 57 0 40 58 40 59 40 60 40 61 40 6240 63 40 64 40

[0462] These results demonstrate that the inventive fractions and theinventive compounds indeed have utility in antineoplastic compositions,and are not toxic in low to medium dosages, with toxicity in higherdosages able to be determined without undue experimentation.

Example 22 Antimicrobial Activity of Cocoa Extracts

[0463] Method A

[0464] A study was conducted to evaluate the antimicrobial activity ofcrude procyanidin extracts from cocoa beans against a variety ofmicroorganisms important in food spoilage or pathogenesis. The cocoaextracts from Example 2, method A were used in the study. An agar mediumappropriate for the growth of each test culture (99 mL) was seeded with1 mL of each cell culture suspension in 0.45% saline (final population10²-10⁴ cfu/mL), and poured into petri dishes. Wells were cut intohardened agar with a #2 cork borer (5 mm diameter). The plates wererefrigerated at 4° C. overnight, to allow for diffusion of the extractinto the agar, and subsequently incubated at an appropriate growthtemperature for the text organism. The results were as follows: SampleZone of Inhibition (mm) Extract Concentration B. B. P. B. (mg/mL)sphericus cereus S. aureus aeruginosa subtilis 0 NI NI NI NI NI 25 NI 12NI 11 NI 250 12 20 19 19 11 500 14 21 21 21 13

[0465] Antimicrobial activity of purified procyanidin extracts fromcocoa beans was demonstrated in another study using the well diffusionassay described above (in Method A) with Staphylococcus aureus as thetext culture. The results were as follows: cocoa extracts: 10 mg/l00 μLdecaffeinated/ detheobrominated acetone extract as in Example 13, methodA 10 mg/100 μL dimer (99% pure) as in Example 14, method D 10 mg/100 μLtetramer (95% pure) as in Example 14, method D 10 mg/100 μL hexamer (88%pure) as in Example 14, method D 10 mg/100 μL octamer/nonamer (92% pure)as in Example 14, method D 10 mg/100 μL monamer & higher (87% pure) asin Example 14, method D Sample Zone of Inhibition (mm) 0.45% saline 0Dimer 33 Tetramer 27 Hexamer 24 0.45% saline 0 Octamer 22 Nonamer 20Decaff./detheo. 26

[0466] Method B

[0467] Crude procyanidin extract as in Example 2, method 2 was added invarying concentrations to TSB (Trypticase Soy Broth) with phenol red(0.08 g/L), The TSB were inoculated with cultures of Salmonellaenteritidis or S. newport (10⁵ cfu/mL), and were incubated for 18 hoursat 35° C. The results were as follows: S. enteritidis S. Newport  0mg/mL + +  50 + + 100 + + 250 + − 500 − − 750 − −

[0468] where +=outgrowth, and −=no growth, as evidenced by the change inbroth culture from red to yellow with acid production. Confirmation ofinhibition was made by plating from TSB tubes onto XLD plates.

[0469] This Example demonstrates that the inventive compounds are usefulin food preparation and preservation.

[0470] This Example further demonstrates that gram negative and grampositive bacterial growth can be inhibited by the inventive compounds.From this, the inventive compounds can be used to inhibit Helicobacterpylori. Helicobacter pylori has been implicated in causing gastriculcers and stomach cancer. Accordingly, the inventive compounds can beused to treat or prevent these and other maladies of bacterial origin.Suitable routes of administration, dosages, and formulations can bedetermined without undue experimentation considering factors well knownin the art such as the malady, and the age, weight, sex, general healthof the subject.

Example 23 Halogen-Free Analytical Separation of Extract

[0471] Procyanidins obtained from Example 2 were partially purified byAnalytical Separation by Halogen-free Normal Phase Chromatography on 100Å Supelcosil LC-Si 5 μm (250×4.6 mm), at a flow rate of 1.0 mL/min, anda column temperature of 37° C. Separations were aided by a lineargradient under the following conditions: (time, %A, %B); (0, 82, 14);(30, 67.6, 28.4); (60, 46, 50). Mobile phase composition was A=30/70 %diethyl ether/Toluene; B=Methanol; and C=acetic acid/water (1:1).Components were detected by UV at 280 nm. A representative elutionprofile is shown in FIG. 28.

Example 24 Effect of Pore Size of Stationary Phase for Normal Phase HPLCSeparation of Procyanidins

[0472] To improve the separation of procyanidins, the use of a largerpore size of the silica stationary phase was investigated. Separationswere effected on Silica-300, 5 μm, 300 Å (250×2.0 mm), or, in thealternative, on Silica-1000, 5 μm, 1000 Å (250×2.0 mm). A lineargradient was employed as mobile phase composition was:A=Dichloromethane; B=Methanol; and C=acetic acid/water (1:1). Componentswere detected by fluorescence, wherein λ_(ex)=276 nm and λ_(em)=316 nm,by UV detector at 280 nm. The flow rate was 1.0 mL/min, and the oventemperature was 37° C. A representative chromatogram from threedifferent columns (100 Å pore size, from Example 13, Method D) is shownin FIG. 29. This shows effective pore size for separation ofprocyanidins.

Example 25 Obtaining Desired Procyanidins Via Manipulating Fermentation

[0473] Microbial strains representative of the succession associatedwith cocoa fermentation were selected from the M&M/Mars cocoa culturecollection. The following isolates were used:

[0474]Acetobacter aceti ATCC 15973

[0475] Lactobacillus sp. (BH 42)

[0476]Candida cruzii (BA 15)

[0477]Saccharomyces cerevisiae (BA 13)

[0478]Bacillus cereus (BE 35)

[0479]Bacillus sphaericus (ME 12)

[0480] Each strain was transferred from stock culture to fresh media.The yeasts and Acetobacter were incubated 72 hours at 26° C. and thebacilli and Lactobacillus were incubated 48 hours at 37° C. The slantswere harvested with 5 mL phosphate buffer prior to use.

[0481] Cocoa beans were harvested from fresh pods and the pulp and testaremoved. The beans were sterilized with hydrogen peroxide (35%) for 20seconds, followed by treatment with catalase until cessation ofbubbling. The beans were rinsed twice with sterile water and the processrepeated. The beans were divided into glass jars and processed accordingto the regimens detailed in the following Table: Fermentation ModelWater Ethanol/acid infusate Fermentation daily daily transfer to dailytransfer bench scale transfer solutions of to fermented model to freshalcohol and acid pulp fermentation in water corresponding to pasteurizedon sterile pulp levels determined each successive coinoculated at eachstage of day of with test a model pulp fermentation strains fermentation

[0482] The bench scale fermentation was performed in duplicate. Alltreatments were incubated as indicated below: Day 1: 26° C. Day 2: 26°C. to 50° C. Day 3: 50° C. Day 4: 45° C. Day 5: 40° C.

[0483] The model fermentation was monitored over the duration of thestudy by plate counts to assess the microbial population and HPLCanalysis of the fermentation medium for the production of microbialmetabolites. After treatment, the beans were dried under a laminar flowhood to a water activity of 0.64 and were roasted at 66° C. for 15 min.Samples were prepared for procyanidin analysis. Three beans pertreatment were ground and defatted with hexane, followed by extractionwith an acetone:water:acetic acid (70:29.5:0.5%) solution. The acetonesolution extract was filtered into vials and polyphenol levels werequantified by normal phase HPLC as in Example 13, method B. Theremaining beans were ground and tasted. The cultural and analyticalprofiles of the model bench-top fermentation process is shown in FIGS.30A-C. The procyanidin profiles of cocoa beans subjected to variousfermentation treatments is shown in FIG. 30D.

[0484] This Example demonstrates that the invention need not be limitedto any particular cocoa genotype; and, that by manipulatingfermentation, the levels of procyanidins produced by a particularTheobroma or Herrania species or their inter or intra species specificcrosses thereof can be modulated, e.g., enhanced.

[0485] The following Table shows procyanidin levels determined inspecimens which are representative of the Theobroma genus and theirinter and intra species specific crosses. Samples were prepared as inExamples 1 and 2 (methods 1 and 2), and analyzed as in Examples 13,method B. This data illustrates that the extracts containing theinventive compounds are found in Theobroma and Herrania species, andtheir intra and inter species specific crosses. Theobroma and HerraniaSpecies Procyanidin Levels ppm (μg/g) in defatted powder Oligomer Mono-Tetra- Penta- Hexa- Hepta- Octa- Deca- SAMPLE mer Dimer Trimer mer mermer mer mer Nonamer mer Undecamer Total T. grandiflorum x 3822 3442 53844074 3146 2080 850 421 348 198 tr⁺ 23,765 T. obovatum 1¹ T. grandiflorumx 3003 4098 5411 3983 2931 1914 1090 577 356 198 tr 23,561 T. obovatum2¹ T. grandiflorum x 4990 4980 7556 5341 4008 2576 1075 598 301 144 tr31,569 T. obovatum 3A¹ T. grandiflorum x 3880 4498 6488 4930 3706 25601208 593 323 174 tr 28,360 T. obavatum 3B¹ T. grandiflorum x 2647 35915328 4240 3304 2380 1506 815 506 249 tr 24,566 T. obovatum 4¹ T.grandiflorum x 2754 3855 5299 3872 2994 1990 1158 629 359 196 88 23,194T. obovatum 6¹ T. grandiflorum x 3212 4134 7608 4736 3590 2274 936 446278 126 ND 23,750 T. obovatum SIN¹ T. obovatum 1¹ 3662 5683 9512 53583858 2454 1207 640 302 144 ND 32,820 T. grandiflorum TEFFE² 2608 21783090 2704 2241 1586 900 484 301 148 tr 16,240 T. grandiflorum TEFFE x4773 4096 5289 4748 3804 2444 998 737 335 156 tr 27,380 T. grandiflorum² T. grandiflorum x 4752 3336 4916 3900 3064 2039 782 435 380 228 ND23,832 T. subincanum ¹ T. obovatum x 3379 3802 5836 3940 2868 1807 814427 271 136 tr 23,280 T. subincanum ¹ T. speciosum x 902 346 1350 217152 120 60 tr tr ND ND 3,147 T. sylvestris ¹ T. microcarpum ² 5694 32502766 1490 822 356 141 tr ND ND ND 14,519 T. cacao, SIAL 659, t0 21,92910,072 10,106 7788 5311 3242 1311 626 422 146 tr 60,753 T. cacao, SIAL659, t24 21,088 9762 9119 7094 4774 2906 1364 608 361 176 tr 57,252 T.cacao, SIAL 659, t48 20,887 9892 9474 7337 4906 2929 1334 692 412 302 tr58,165 T. cacao, SIAL 659, t96 9552 5780 5062 3360 2140 1160 464 254 138tr ND 27,910 T. cacao, SIAL 659, t120 8581 4665 4070 2527 1628 888 326166 123 tr ND 22,974 Pod Rec. 10/96, 869 1295 545 347 175 97 tr *ND ND3329 Herrania mariae Sample Rec. prior to 10/96, 130 354 151 131 116 51tr ND ND 933 Herrania mariae

Example 26 Effect of Procyanidins on NO

[0486] Method A

[0487] The purpose of this study is to establish the relationshipbetween procyanidins (as in Example 14, method D) and NO, which is knownto induce cerebral vascular dilation. The effects of monomers and higheroligomers, in concentrations ranging from 100 μg/mL to 0.1 μg/mL, on theproduction of nitrates (the catabolites of NO), from HUVEC (humanumbilical vein endothelial cells) is evaluated. HUVEC (from Clonetics)is investigated in the presence or absence of each procyanidin for 24 to48 hours. At the end of the experiments, the supernatants are collectedand the nitrate content determined by calorimetric assay. In separateexperiments, HUVEC is incubated with acetylcholine, which is known toinduce NO production, in the presence or absence of procyanidins for 24to 48 hours. At the end of the experiments, the supernatants arecollected and nitrate content is determined by calorimetric assay. Therole of NO is ascertained by the addition of nitroarginine or(1)-N-methyl arginine, which are specific blockers of NO synthase.

[0488] Method B. Vasorelaxation of Phenylephrine-Induced Contracted RatArtery

[0489] The effects of each of the procyanidins (100 μg/mL to 0.1 μg/mLon the rat artery is the target for study of vasorelaxation ofphenylephrine-induced contracted rat artery. Isolated rat artery isincubated in the presence or absence of procyanidins (as in Example 14,method D) and alteration of the muscular tone is assessed by visualinspection. Both contraction or relaxation of the ray artery isdetermined. Then, using other organs, precontraction of the isolated ratartery is induced upon addition of epinephrine. Once the contraction isstabilized, procyanidins are added and contraction or relaxation of therat artery is determined. The role of NO is ascertained by the additionof nitroarginine or (1)-N-methyl arginine. The acetylcholine-inducedrelaxation of NO, as it is effected by phenylephrine-precontracted rataorta is shown in FIG. 31.

[0490] Method C. Induction of Hypotension in the Rat

[0491] This method is directed to the effect of each procyanidin (as inExample 14, method D) on blood pressure. Rats are instrumented in orderto monitor systolic and diastolic blood pressure. Each of theprocyanidins are injected intravenously (dosage range=100-0.1 μg/kg),and alteration of blood pressure is assessed. In addition, the effect ofeach procyanidin on the alteration of blood pressure evoked byepinephrine is determined. The role of NO is ascertained by the additionof nitroarginine or (1)-N-methyl arginine.

[0492] These studies, together with next Example, illustrate that theinventive compounds are useful in modulating vasodilation, and arefurther useful with respect to modulating blood pressure or addressingcoronary conditions, and migraine headache conditions.

Example 27 Effects of Cocoa Polyphenols on Satiety

[0493] Using blood glucose levels as an indicator for the signal eventswhich occur in vivo for the regulation of appetite and satiety, a seriesof simple experiments were conducted using a healthy male adultvolunteer age 48 to determine whether cocoa polyphenols would modulateglucose levels. Cocoa polyphenols were partially purified from Braziliancocoa beans according to the methods described by Clapperton et al.(1992). This material contained no caffeine or theobromine. Fastingblood glucose levels were analyzed on a timed basis after ingestion of10 fl. oz of Dexicola 75 (caffeine free) Glucose tolerance test beverage(Curtin Matheson 091-421) with and without 75 mg cocoa polyphenols. Thislevel of polyphenols represented 0.1% of the total glucose of the testbeverage and reflected the approximate amount that would be present in astandard 100 g chocolate bar. Blood glucose levels were determined byusing the Accu-Chek III blood glucose monitoring system (BoehringerMannheim Corporation). Blood glucose levels were measured beforeingestion of test beverage, and after ingestion of the test beverage atthe following timed intervals: 15, 30, 45, 60, 75, 90, 120 and 180minutes. Before the start of each glucose tolerance test, high and lowglucose level controls were determined. Each glucose tolerance test wasperformed in duplicate. A control test solution containing 75 mg cocoapolyphenols dissolved in 10 fl. oz. distilled water (no glucose) wasalso performed.

[0494] Table 11 below lists the dates and control values obtained foreach glucose tolerance experiment performed in this study. FIG. 32represents plots of the average values with standard deviations of bloodglucose levels obtained throughout a three hour time course. It isreadily apparent that there is a substantial increase in blood sugarlevels was obtained after ingestion of a test mixture containing cocoapolyphenols. The difference between the two principal glucose toleranceprofiles could not be resolved by the profile obtained after ingestionof a solution of cocoa polyphenols alone. The addition of cocoapolyphenols to the glucose test beverage raised the glucose toleranceprofile significantly. This elevation in blood glucose levels is withinthe range considered to be mildly diabetic, even though the typicalglucose tolerance profile was considered to be normal (Davidson, I. etal., Eds. Todd-Sandford Clinical Diagnosis by Laboratory Methods 14thedition; W. B. Saunders Co.; Philadelphia, Pa. 1969 Ch. 10, pp. 550-9).This suggests that the difference in additional glucose was released tothe bloodstream, from the glycogen stores, as a result of the inventivecompounds. Thus, the inventive compounds can be used to modulate bloodglucose levels when in the presence of sugars. TABLE 11 GlucoseTolerance Test Dates and Control Results HIGH LOW WEEK 1 DESCRIPTIONCONTROL^(a) CONTROL^(b) 0 Glucose Tolerance 265 mg/dL 53 mg/dL 1 GlucoseTolerance 310 68 with 0.1% polyphenols 2 Glucose Tolerance 315 66 4Glucose Tolerance 325 65 with 0.1% polyphenols 5 0.1% polyphenols 321 66

[0495] The subject also experienced a facial flush (erythema) andlightheadedness following ingestion of the inventive compounds,indicating modulation of vasodilation.

[0496] The data presented in Tables 12 and 13 illustrates the fact thatextracts of the invention pertaining to cocoa raw materials andcommercial chocolates, and inventive compounds contained therein can beused as a vehicle for pharmaceutical, veterinary and food sciencepreparations and applications. TABLE 13 Procyanidin Levels in CommercialChocolates μg/g Heptamers and Sample Monomers Dimers Trimers TetramersPentamers Hexamers Higher Total Brand 1 366 166 113 59 56 23 18 801Brand 2 344 163 111 45 48 ND* ND 711 Brand 3 316 181 100 41 40 7 ND 685Brand 4 310 122 71 27 28 5 ND 563 Brand 5 259 135 90 46 29 ND ND 559Brand 6 308 139 91 57 47 14 ND 656 Brand 7 196 98 81 58 54 19 ND 506Brand 8 716 472 302 170 117 18 ND 1,795 Brand 9 1,185 951 633 298 173 2521 3,286 Brand 10 1,798 1,081 590 342 307 93 ND 4,211 Brand 11 1,101 746646 372 347 130 75 3,417 Brand 12 787 335 160 20 10 8 ND 1,320

[0497] TABLE 14 Procyanidin Levels in Cocoa Raw Materials μg/g Heptamersand Sample Monomers Dimers Trimers Tetramers Pentamers Hexamers HigherTotal Unfermented 13,440 6,425 6,401 5,292 4,236 3,203 5,913 44,910Fermented 2,695 1,538 1,362 740 470 301 277 7,383 Roasted 2,656 1,597921 337 164 ND* ND 5,675 Choc. Liquor 2,805 1,446 881 442 184 108 ND5,866 Cocoa Hulls 114 53 14 ND ND ND ND 181 Cocoa Powder 1% Fat 506 287112 ND ND ND ND 915 Cocoa Powder 11% Fat 1,523 1,224 680 46 ND ND ND3,473 Red Dutch Cocoa 1,222 483 103 ND ND ND ND 1,808 Powder, pH 7.4,11% fat Red Dutch Cocoa 168 144 60 ND ND ND ND 372 Powder, pH 8.2, 23%fat

Example 28 The Effect of Procyanidins on Cyclooxygenase 1 & 2

[0498] The effect of procyanidins on cyclooxygenase 1 & 2 (COX1/COX2)activities was assessed by incubating the enzymes, derived from ramseminal vesicle and sheep placenta, respectively, with arachidonic acid(5 μM) for 10 minutes at room temperature, in the presence of varyingconcentrations of procyanidin solutions containing monomer to decamerand procyanidin mixture. Turnover was assessed by using PGE2 EIA kitsfrom Interchim (France). Indomethacin was used as a reference compound.The results are presented in the following Table, wherein the IC₅₀values are expressed in units of μM (except for S11, which represents aprocyanidin mixture prepared from Example 13, Method A and where thesamples S1 to S10 represent sequentially procyanidin oligomers (monomerthrough decamer) as in Example 14, Method D, and IC₅₀ is expressed inunits of mg/mL). IC₅₀ COX-1 IC₅₀ COX-2 RATIO IC₅₀ SAMPLE # (*) (*)COX2/COX1 1 0.074 0.197 2.66 2 0.115 0.444 3.86 3 0.258 0.763 2.96 40.154 3.73 24.22 5 0.787 3.16 4.02 6 1.14 1.99 1.75 7 1.89 4.06 2.15 82.25 7.2 3.20 9 2.58 2.08 0.81 10 3.65 3.16 0.87 11 0.0487 0.0741 1.52Indomethacin 0.599 13.5 22.54

[0499] The results of the inhibition studies are presented in FIGS. 33 Aand B, which shows the effects of Indomethacin on COX1 and COX2activities. FIGS. 34 A and B shows the correlation between the degree ofpolymerization of the procyanidin and IC₅₀ with COX1 and COX2; FIG. 35shows the correlation between IC₅₀ values on COX1 and COX2. And, FIGS.36 A through Y show the IC₅₀ values of each sample (S1-S11) with COX1and COX2.

[0500] These results indicate that the inventive compounds haveanalgesic, anti-coagulant, and anti-inflammatory utilities. Further,COX2 has been linked to colon cancer. Inhibition of COX2 activity by theinventive compounds illustrates a plausible mechanism by which theinventive compounds have antineoplastic activity against colon cancer.

[0501] COX1 and COX2 are also implicated in the synthesis ofprostaglandins. Thus, the results in this Example also indicate that theinventive compounds can modulate renal functions, immune responses,fever, pain, mitogenesis, apoptosis, prostaglandin synthesis, ulceration(e.g., gastric), and reproduction. Note that modulation of renalfunction can affect blood pressure; again implicating the inventivecompounds in modulating blood pressure, vasodilation, and coronaryconditions (e.g., modulation of angiotensin, bradykinin).

[0502] Reference is made to Seibert et al., PNAS USA 91:12013-12017(December, 1994), Mitchell et al., PNAS USA 90:11693-11697 (December1994), Dewitt et al., Cell 83:345-348 (Nov. 3, 1995), Langenbach et al.,Cell 83:483-92 (Nov. 3, 1995) and Sujii et al., Cell 83:493-501 (Nov. 3,1995), Morham et al., Cell 83:473-82 (Nov. 3, 1995).

[0503] Reference is further made to Examples 9, 26, and 27. In Example9, the anti-oxidant activity of inventive compounds is shown. In Example26, the effect on NO is demonstrated. And, Example 27 provides evidenceof a facial vasodilation. From the results in this Example, incombination with Examples 9, 26 and 27, the inventive compounds canmodulate free radical mechanisms driving physiological effects.Similarly, lipoxygenase mediated free radical type reactionsbiochemically directed toward leukotriene synthesis can be modulated bythe inventive compounds, thus affecting subsequent physiological effects(e.g., inflammation, immune response, coronary conditions, carcinogenicmechanisms, fever, pain, ulceration).

[0504] Thus, in addition to having analgesic properties, there may alsobe a synergistic effect by the inventive compounds when administeredwith other analgesics. Likewise, in addition to having antineoplasticproperties, there may also be a synergistic effect by the inventivecompounds when administered with other antineoplastic agents.

Example 29 Circular Dichroism/Study of Procyanidins

[0505] CD studies were undertaken in an effort to elucidate thestructure of purified procyanidins as in Example 14, Method D. Thespectra were collected at 25° C. using CD spectrum software AVIV 60DSV4.1f.

[0506] Samples were scanned from 300 nm to 185 nm, every 1.00 nm, at1.50 nm bandwidth. Representative CD spectra are shown in FIGS. 43Athrough G, which show the CD spectra of dimer through octamer.

[0507] These results are indicative of the helical nature of theinventive compounds.

Example 30 Inhibitory Effects of Cocoa Procyanidins on Helicobacterpylori and Staphylococcus aureus

[0508] A study was conducted to evaluate the antimicrobial activity ofprocyanidin oligomers against Helicobacter pylori and Staphylococcusaureus. Pentamer enriched material was prepared as described in Example13, Method A and analyzed as described in Example 14, Method C, where89% was pentamer, and 11% was higher oligomers (n is 6 to 12). Purifiedpentamer (96.3%) was prepared as described in Example 14, Method D.

[0509]Helicobacter pylori and Staphylococcus aureus were obtained fromthe American Type Culture Collection (ATCC). For H. pylori, the vial wasrehydrated with 0.5 mL Trypticase Soy broth and the suspensiontransferred to a slant of fresh TSA containing 5% defibrinated sheepblood. The slant was incubated at 37° C. for 3 to 5 days undermicroaerophilic conditions in anaerobic jars (5 to 10% carbon dioxide;CampyPakPlus, BBL). When good growth was established in the pool ofbroth at the bottom of the slant, the broth was used to inoculateadditional slants of TSA with sheep blood. Because viability decreasedwith continued subculturing, the broth harvested from the slants waspooled and stored at −80° C. Cultures for assay were used directly fromthe frozen vials. The S. aureus culture was maintained on TSA slants andtransferred to fresh slants 24 h prior to use.

[0510] A cell suspension of each culture was prepared (H. pylori, 10⁸ to10⁹ cfu/mL; S. aureus 10⁶ to 10⁷ cfu/mL) and 0.5 mL spread onto TSAplates with 5% sheep blood. Standard assay disks (Difco) were dippedinto filter sterilized, serial dilutions of pentamer (23 mg/mL intosterile water). The test disks and the blank control disks (sterilewater) were placed on the inoculated plates. Control disks containing 80ug metronidazole (inhibitory to H. pylori) or 30 ug vancomycin(inhibitory to S. aureus) (BBL Sensidiscs) were also placed on theappropriate set of plates. The H. pylori inoculated plates wereincubated under microaerophilic conditions. The S. aureus set wasincubated aerobically. Zones of inhibition were measured followingoutgrowth. TABLE 14 Bioassays with pentamer against Helicobacter pyloriand Staphylocuccus aureus Pentamer Enriched S. aureus H. pylori Fraction(mg/ml) Inhibition (mm) Inhibition (mm)  0 NI NI  15  0 10  31 10 10  6211 11 125 13 13 250 15 13 Vancomycin 15 — standard Metronidazole — 11standard 96% pure pentamer 15 11

Example 31 NO Dependent Hypotension in the Guinea Pig

[0511] The effect of five cocoa procyanidin fractions on guinea pigblood pressure were investigated. Briefly, guinea pigs (approximately400 g body weight; male and female) were anesthetized upon injection of40 mg/kg sodium pentobarbital. The carotid artery was cannulated formonitoring of the arterial blood pressure. Each of the five cocoaprocyanidin fractions was injected intravenously (dose range 0.1mg/kg-100 mg/kg) through the jugular vein. Alterations of blood pressurewere recorded on a polygraph. In these experiments, the role of NO wasascertained by the administration of L-N-methylarginine (1 mg/kg) tenminutes prior to the administration of cocoa procyanidin fractions.

[0512] Cocoa procyanidin fractions were prepared and analyzed accordingto the procedures described in U.S. Pat. No. 5,554,645, herebyincorporated herein by reference. Fraction A: Represents a preparativeHPLC fraction comprised of monomers-tetramers. HPLC analysis revealedthe following composition: Monomers 47.2% Dimers 23.7 Trimers 18.7Tetramers 10.3 Fraction B: Represents a preparative HPLC fractioncomprised of pentamer-decamers. HPLC analysis revealed the followingcomposition: Pentamers 64.3% Hexamers 21.4 Heptamers  7.4 Octamers  1.9Nonamers  0.9 Decamers  0.2 Fraction C: Represents an enriched cocoaprocyanidin fraction used in the preparation of Fractions A and B(above). HPLC analysis revealed the following composition: Monomers34.3% Dimers 17.6 Trimers 16.2 Tetramers 12.6 Pentamers  8.5 Hexamers 5.2 Heptamers  3.1 Octamers  1.4 Nonamers  0.7 Decamers  0.3 FractionD: Represents a procyanidin extract prepared from a milk chocolate. HPLCanalysis revealed a composition similar to that listed in the Table 12for Brand 8. Additionally, caffeine 10% and theobromine 6.3% werepresent. Fraction E: Represents a procyanidin extract prepared from adark chocolate prepared with alkalized liquor. HPLC analysis revealed acomposition similar to that listed in the Table 12 for Brand 12.Additionally, caffeine 16.0% and theobromine 5.8% were present.

[0513] In three separate experiments, the effects of administering 10mg/kg cocoa procyanidin fractions on arterial blood pressure ofanesthetized guinea pigs was investigated. Upon intravenous injection,procyanidin fractions A and E evoked a decrease in blood pressure ofabout 20%. This decrease was only marginally different from thatobtained from a solvent (DMSO) control (15±5%, n=5). In contrast,procyanidin fractions B, C and D (10 mg/kg) induced marked decreases inblood pressure, up to 50-60% for C. In these experiments the order ofhypotensive effect was as follows: C>B>D>>A=E.

[0514] Typical recordings of blood pressure elicited after injection ofprocyanidin fractions appear in FIG. 50A for fraction A and FIG. 50B forfraction C. FIG. 51 illustrates the comparative effects on bloodpressure by these fractions.

[0515] The possible contribution of NO in the hypotension in the guineapig induced by administration of fraction C was analyzed usingL-N-methyl arginine (LNMMA). This pharmacological agent inhibits theformation of NO by inhibiting NO synthase. L-NMMA was administered atthe dose of 1 mg/kg, ten minutes prior to injection of the cocoaprocyanidin fractions. Treatment of the animals with L-NMMA completelyblocked the hypotension evoked by the procyanidin fraction C. Indeed,following treatment with this inhibitor, the alterations of bloodpressure produced by fraction C were similar to those noted with solventalone.

Example 32 Effect of Cocoa Procyanidin Fractions on NO Production inHuman Umbilical Vein Endothelial Cells

[0516] Human umbilical vein endothelial cells (HUVEC) were obtained fromClonetics and cultures were carried out according to the manufacturer'sspecifications. HUVEC cells were seeded at 5,000 cells/cm in 12-wellplates (Falcon). After the third passage under the same conditions, theywere allowed to reach confluence. The supernatant was renewed with freshmedium containing defined concentrations of bradykinin (25, 50 and100nM) or cocoa procyanidin fractions A-E (100 μg/mL) as described inexample 31. The culture was continued for 24 hr. and the cell freesupernatants were collected and stored frozen prior to assessment of NOcontent as described below. In selected experiments, the NO synthase(NOS) antagonist, Nω-nitro-L-arginine methyl ester (L-NAME, 10 μM) wasadded to assess the involvement of NOS in the observed NO production.

[0517] HUVEC NO production was estimated by measuring nitriteconcentration in the culture supernatant by the Griess reaction. Griessreagent was 1% sulfanilamide, 0.1% N-(1-naphthyl)-ethylenediaminedihydrochloride. Briefly, 50 μL aliquots were removed from the varioussupernatants in quadruplicate and incubated with 150 μL of the Griessreagent. The absorbency at 540 nm was determined in a multiscan(Labsystems Multiskans MCC/340) apparatus. Sodium nitrite was used atdefined concentrations to establish standard curves. The absorbency ofthe medium without cells (blank) was subtracted from the value obtainedwith the cell containing supernatants.

[0518]FIG. 53 illustrates the effect of bradykinin on NO production byHUVEC where a dose dependent release of NO was observed. The inhibitorL-NAME completely inhibited the bradykinin induced NO release.

[0519]FIG. 54 illustrates the effect of the cocoa procyanidin fractionson NO production by HUVEC cells. Fractions B, C and D induced a moderatebut significant amount of NO production by HUVEC. By far, Fraction C wasthe most efficient fraction to induce NO formation as assessed by theproduction of nitrites, while Fraction E was nearly ineffective. Theeffect of Fraction C on NO production was dramatically reduced in thepresence of L-NAME. Interestingly, Fractions B, C and D contained higheramounts of procyanidin oligomers than Fractions A and E. Adistinguishing difference between Fractions D and E was that E wasprepared from a dark chocolate which used alkalized cocoa liquor as partof the chocolate recipe. Alkalization leads to a base catalyzedpolymerization of procyanidins which rapidly depletes the levels ofthese compounds. An analytical comparison of procyanidin levels found inthese types of chocolate appear in the Table 12, where Brand 12 is adark chocolate prepared with alkalized cocoa liquor and Brand 11 is atypical milk chocolate. Thus, extracts obtained from milk chocolatescontain high proportions of procyanidin oligomers which are capable ofinducing NO. The addition of the NO inhibitor L-NMMA to the Fraction Csample clearly led to the inhibition of NO. The results obtained fromthe procyanidin fractions were consistent to those observed with thebradykinin induced NO experiment (see FIG. 53).

[0520] As in the case of the HUVEC results, cocoa procyanidin fraction Celicited a major hypotensive effect in guinea pigs, whereas fractions Aand E were the least effective. Again, the presence of high molecularweight procyanidin oligomers were implicated in the modulation of NOproduction.

Example 33 Effect of Cocoa Procyanidin Fractions on Macrophage NOProduction

[0521] Fresh, human heparinized blood (70 mL) was added with an equalvolume of phosphate buffer saline (PBS) at room temperature. AFicoll-Hypaque solution was layered underneath the blood-PBS mixtureusing a 3 mL Ficoll-Hypaque to 10 mL blood-PBS dilution ratio. The tubeswere centrifuged for 30 minutes at 2,000 rpm at 18-20° C. The upperlayer containing plasma and platelets was discarded. The mononuclearcell layer was transferred to another centrifuge tube and the cells werewashed 2× in Hanks balanced saline solution. The mononuclear cells wereresuspended in complete RPMI 1640 supplemented with 10% fetal calfserum, counted and the viability determined by the trypan blue exclusionmethod. The cell pellet was resuspended in complete RPMI 1640supplemented with 20% fetal calf serum to a final concentration of 1×10⁶cells/mL. Aliquots of the cell suspension were plated into a 96 wellculture plate and rinsed 3× with RPMI 1640 supplemented with 10% fetalcalf serum and the nonadherent cells (lymphocytes) were discarded.

[0522] These cells were incubated for 48 hours in the presence orabsence of five procyanidin fractions described in Example 31. At theend of the incubation period, the culture media were collected,centrifuged and cell free supernatants were stored frozen for nitrateassay determinations.

[0523] Macrophage NO production was determined by measuring nitriteconcentrations by the Greiss reaction. Greiss reagent was 1%sulfanilamide, 0.1% N-(1-naphthyl)ethylenediamine dihydrochloride.Briefly, 50 μL aliquots were removed from the supernatants inquadruplicate and incubated with 150 μL of the Greiss reagent. Theabsorbency at 540 nm was determined in a multiscan (LabsystemsMultiskans MCC/340) apparatus. Sodium nitrite was used at definedconcentrations to establish standard curves. The absorbency of themedium without cells (blank) was subtracted from the value obtained withthe cell containing supernatants.

[0524] In a separate experiment, macrophages were primed for 12 hours inthe presence of 5 U/mL gamma-interferon and then stimulated with 10μg/mL LPS for the next 36 hours in the presence or absence of 100 μg/mLof the five procyanidin fractions.

[0525]FIG. 55 indicates that only procyanidin fraction C, at 100 μg/mL,could induce NO production by monocytes/macrophages. Basal NO productionby these cells was undetectable and no nitrite could be detected in anyof the cocoa procyanidin fractions used at 100 μg/mL. FIG. 56 indicatesthat procyanidin fractions A and D enhanced LPS-induced NO production byT-interferon primed monocytes/macrophages. Procyanidin fraction C wasmarginally effective, since LPS-stimulated monocytes/macrophagescultured in the absence of procyanidin fractions produced only 4μmole/10⁵ cells/48 hours. T-Interferon alone was ineffective in inducingNO.

[0526] Collectively, these results demonstrate that mixtures of theinventive compounds used at specific concentrations are capable ofinducing monocyte/macrophage NO production both independent anddependent of stimulation by LPS or cytokines.

[0527] From the foregoing, it is clear that the extract and cocoapolyphenols, particularly the inventive compounds, as well as thecompositions, methods, and kits, of the invention have significant andnumerous utilities.

[0528] The antineoplastic utility is clearly demonstrated by the in vivoand in vitro data herein and shows that inventive compounds can be usedinstead of or in conjunction with conventional antineoplastic agents.

[0529] The inventive compounds have antioxidant activity like that ofBHT and BHA, as well as oxidative stability. Thus, the invention can beemployed in place of or in conjunction with BHT or BHA in knownutilities of BHA and BHT, such as an antioxidant, for instance, anantioxidant; food additive.

[0530] The invention can also be employed in place of or in conjunctionwith topoisomerase II-inhibitors in the presently known utilitiestherefor.

[0531] The inventive compounds can be used in food preservation orpreparation, as well as in preventing or treating maladies of bacterialorigin. Simply the inventive compounds can be used as an antimicrobial.

[0532] The inventive compounds can also be used as a cyclo-oxygenaseand/or lipoxygenase, NO or NO-synthase, or blood or in vivo glucosemodulator, and are thus useful for treatment or prevention or modulationof pain, fever, inflammation coronary conditions, ulceration,carcinogenic mechanisms, vasodilation, as well as an analgesic,anti-coagulant anti-inflammatory and an immune response modulator.

[0533] Further, the invention comprehends the use of the compounds orextracts as a vehicle for pharmaceutical preparations. Accordingly,there are many compositions and methods envisioned by the invention. Forinstance, antioxidant or preservative compositions, topoisomeraseII-inhibiting compositions, methods for preserving food or any desireditem such as from oxidation, and methods for inhibiting topoisomeraseII. The compositions can comprise the inventive compounds. The methodscan comprise contacting the food, item or topoisomerase II with therespective composition or with the inventive compounds. Othercompositions, methods and embodiments of the invention are apparent fromthe foregoing.

[0534] In this regard, it is mentioned that the invention is from anedible source and, that the activity in vitro can demonstrate at leastsome activity in vivo; and from the in vitro and in vivo data herein,doses, routes of administration, and formulations can be obtainedwithout undue experimentation

Example 34 Micellar Electrokinetic Capillary Chromatography of CocoaProcyanidins

[0535] A rapid method was developed using micellar electrokineticcapillary chromatography (MECC) to separate procyanidin oligomers. Themethod is a modification of that reported by Delgado et al., 1994. TheMECC method requires only 12 minutes to achieve the same separation asthat obtained by a 70 minute normal phase HPLC analysis. FIG. 57represents a MECC separation of cocoa procyanidins obtained by Example2.

[0536] MECC Conditions

[0537] The cocoa procyanidin extract was prepared by the methoddescribed in Example 2 and dissolved at a concentration of 1 mg/mL inMECC buffer consisting of 200 mM boric acid, 50 mM sodium dodecylsulfate (electrophoresis pure) and NaOH to adjust to pH=8.5.

[0538] The sample was passed through a 0.45 um filter andelectrophoresed using a Hewlett Packard HP-3D CZE System operated at thefollowing conditions:

[0539] Inlet buffer: Run buffer as described above

[0540] Outlet buffer: Run buffer as described above

[0541] Capillary: 50 cm×75 um i.d. uncoated fused silica

[0542] Detection: 200 nm, with Diode Array Detector

[0543] Injection: 50 mBar for 3 seconds (150 mBar sec)

[0544] Voltage: 6 watts

[0545] Amperage: System limit (<300 uA)

[0546] Temperature: 25° C.

[0547] Capillary Condition: 5 min flush with run buffer before and aftereach run.

[0548] This method can be modified by profiling temperature, pressure,and voltage parameters, as well as including organic modifiers andchiral selective agents in the run buffer.

Example 35 MALDI-TOF/MS Analysis of Procyanidin Oligomers with MetalSalt Solutions

[0549] A series of MALDI-TOF/MS analyses were performed on trimerscombined with various metal salt solutions to determine whether cationadducts of the oligomer could be detected. The significance of theexperiment was to provide evidence that the procyanidin oligomers play aphysiological role in vitro and in vivo by sesquestering or deliveringmetal cations important to physiological processes and disease.

[0550] The method used was as described in Example 15. Briefly, 2 uL of10 mM solutions of zinc sulfate dihydrate, calcium chloride, magnesiumsulfate, ferric chloride hexahydrate, ferrous sulfate heptahydrate, andcupric sulfate were individually combined with 4 uL of a trimer (10mg/mL) purified to apparent homogeneity as described in Example 14, and44 uL of DHB added.

[0551] The results (FIGS. 58A-F) showed [Metal−Trimer+H]⁺ ions forcopper and iron (ferrous and ferric) whose m/z values matched ±1 amustandard deviation value for the theoretical calculated masses. The[Metal−Trimer+H]⁺ masses for calcium and magnesium could not beunequivocally resolved from the [Metal-Trimer+H]⁺ masses for sodium andpotassium, whose m/z values were within the ±1 amu standard deviationvalues. No [Zn⁺²−Trimer+H]⁺ ion could be detected. Since some of thesecations are multi-valent, the possibility for multimetal-oligomer(s)ligand species and/or metal-multioligomer species were possible.However, scanning for these adducts at their predicted masses provedunsuccessful.

[0552] The results shown above for copper, iron, calcium, magnesium andzinc may be used as general teachings for subsequent analysis of thereaction between other metal ions and the inventive compounds, takinginto account such factors as oxidation state and the relative positionin the periodic table of the ion in question.

Example 36 MALDI-TOF/MS Analysis of High Molecular Weight ProcyanidinOligomers

[0553] An analytical examination was made on GPC eluants associated withhigh molecular weight procyanidin oligomers as prepared in Example 3,Method A. The objective was to determine whether procyanidin oligomerswith n>12 were present. If present, these oligomers represent additionalcompounds of the invention. Adjustments to existing methods ofisolation, separation and purification embodied in the invention can bemade to obtain these oligomers for subsequent in vitro and in vivoevaluation for anti-cancer, anti-tumor or antineoplastic activity,antioxidant activity, inhibit DNA topoisomerase II enzyme, inhibitoxidative damage to DNA, and have antimicrobial, NO or NO-synthase,apoptosis, platelet aggregation, and blood or in vivo glucose modulatingactivities, as well as efficacy as non-steroidal antiinflammatoryagents.

[0554]FIG. 59 represents a MALDI-TOF mass spectrum of the GPC eluantsample described above. The [M+Na)⁺ and/or [M+K]⁺ and/or [M+2Na]⁺ ionscharacterizing procyanidin oligomers representative of tetramers throughoctadecamers are clearly evident.

[0555] It was learned that an acid and heat treatment will cause thehydrolysis of procyanidin oligomers. Therefore, the inventioncomprehends the controlled hydrolysis of high molecular weightprocyanidin oligomers (e.g. where n is 13 to 18) as a method to preparelower molecular weight procyanidin oligomers (e.g. where n is 2 to 12).

Example 37 Dose Response Relationships of Procyanidin Oligomers andCanine and Feline Cell Lines

[0556] The dose respone effects of procyanidin oligomers were evaluatedagainst several canine and feline cell lines obtained from the WalthamCenter for Pet Nutrition, Waltham on-the-Wolds, Melton Mowbray,Leicestershire, U.K. These cell lines were

[0557] Canine normal kidney GH cell line;

[0558] Canine normal kidney MDCK cell line;

[0559] Feline normal kidney CRFK cell line; and

[0560] Feline lymphoblastoid FeA cell line producing leukemia viruswhich were cultured under the conditions described in Example 8, MethodA.

[0561] Monomers and procyanidin oligomers, where n is 2 to 10 werepurified as described in Example 14, Method D. The oligomers were alsoexamined by analytical normal phase HPLC as described in Example 14,Method C, where the following results were obtained. Procyanidin %Purity by HPLC Monomers 95.4 Dimers 98.0 Trimers 92.6 Tetramers 92.6Pentamers 93.2 Hexamers 89.2 (Contains 4.4% pentamers) Heptamers 78.8(Contains 18.0% hexamers) Octamers 76.3 (contains 16.4% heptamers)Nonamers 60.3 (Contains 27.6% octamers) Decamers 39.8 (Contains 22.2%nonamers, 16.5% octamers, and 13.6% heptamers)

[0562] In those cases where the purity of the oligomer is <90%, methodsembodied in the invention are used for their repurification.

[0563] Each cell line was dosed with monomers and each procyanidinoligomer at 10 ug/mL, 50 ug/mL and 100 ug/mL and the results shown inFIGS. 60-63. As shown in the Figures, high dose (100 ug/mL)administration of individual oligomers produced similar inhibitoryeffects on the feline FeA lymphoblastoid and feline normal kidney CRFKcell lines. In these cases, cytotoxicity appeared with the tetramer, andincreasingly higher oligomers elicited increasingly higher cytotoxiceffects. By contrast, high dose (100 ug/mL) administration of individualoligomers to canine GH and MDCK normal kidney cell lines required ahigher oligomer to initiate the appearance of cytotoxicity. For thecanine GH normal kidney cell line, cytotoxicity appeared with thepentamer. For the canine MDCK normal kidney cell line, cytotoxicityappeared with the hexamer. In both of these cases, the administration ofhigher oligomers produced increasing levels of cytotoxicity.

Example 38 Tablet Formulations

[0564] A tablet formulation was prepared using cocoa solids obtained bymethods described in U.S. application Ser. No. 08/709,406 filed Sep. 6,1996, hereby incorporated herein by reference. Briefly, this ediblematerial is prepared by a process which enhances the natural occurrenceof the compounds of the invention in contrast to their levels found intraditionally processed cocoa, such that the ratio of the initial amountof the compounds of the invention found in the unprocessed bean to thatobtained after processing is less than or equal to 2. For simplicity,this cocoa solids material is designated herein as CP-cocoa solids. Theinventive compound or compounds, e.g., in isolated and/or purified formmay be used in tablets as described in this Example, instead of or incombination with CP-cocoa solids.

[0565] A tablet formula comprises the following (percentages expressedas weight percent): CP-cocoa solids  24.0% 4-Fold Natural vanillaextract (Bush Boake Allen)  1.5% Magnesium stearate (dry lubricant)(AerChem, Inc.)  0.5% Dipac tabletting sugar (Amstar Sugar Corp.)  37.0%Xylitol (American Xyrofin, Inc.)  37.0% 100.0%

[0566] The CP-cocoa solids and vanilla extract are blended together in afood processor for 2 minutes. The sugars and magnesium stearate aregently mixed together, followed by blending in the CP-cocoasolids/vanilla mix. This material is run through a Manesty Tablet Press(B3B) at maximum pressure and compaction to produce round tablets (15mm×5 mm) weighing 1.5-1.8 gram. Another tablet of the above mentionedformula was prepared with a commercially available low fat natural cocoapowder (11% fat) instead of the CP-cocoa solids (11% fat). Both tabletformulas produced products having acceptable flavor characteristics andtexture attributes.

[0567] An analysis of the two tablet formulas was performed using theprocedures described in Example 4, Method 2. In this case, the analysisfocused on the concentration of the pentamer and the total level ofmonomers and compounds of the invention where n is 2 to 12 which arereported below. pentamer total Tablet pentamer total (ug/l.8 g (ug/l.8 gsample (ug/g) (ug/g) serving) serving) tablet with CP⁻ 239 8,277 43014,989 cocoa solids tablet with ND   868 ND   1563 commercial low fatcocoa powder

[0568] The data clearly showed a higher level of pentamer and totallevel of compounds of the invention in the CP-cocoa solids tablet thanin the other tablet formula. Thus, tablet formulas prepared withCP-cocoa solids are an ideal delivery vehicle for the oraladministration of compounds of the invention, for pharmaceutical,supplement and food applications.

[0569] The skilled artisan in this area can readily prepare other tabletformulas covering a wide range of flavors, colors, excipients, vitamins,minerals, OTC medicaments, sugar fillers, UV protectants (e.g., titaniumdioxide, colorants, etc.), binders, hydrogels, and the like except forpolyvinyl pyrrolidone which would irreversibly bind the compounds of theinvention or combination of compounds. The amount of sugar fillers maybe adjusted to manipulate the dosages of the compounds of the inventionor combination of compounds.

[0570] Many apparent variations of the above are self-evident andpossible without departing from the spirit and scope of the example.

Example 39 Capsule Formulations

[0571] A variation of Example 38 for the oral delivery of the compoundsof the invention is made with push-fit capsules made of gelatin, as wellas soft sealed capsules made of gelatin and a plasticizer such asglycerol. The push-fit capsules contain the compound of the invention orcombination of compounds or CP-cocoa solids as described in Examples 38and 40 in the form of a powder which can be optionally mixed withfillers such as lactose or sucrose to manipulate the dosages of thecompounds of the invention. In soft capsules, the compound of theinvention or combination of compounds or CP-cocoa solids are suspendedin a suitable liquid such as fatty oils or cocoa butter or combinationstherein. Since an inventive compound or compounds may belight-sensitive, e.g., sensitive to UV, a capsule can contain UVprotectants such as titanium dioxide or suitable colors to protectagainst UV. The capsules can also contain fillers such as thosementioned in the previous Example.

[0572] Many apparent variations of the above are self-evident andpossible to one skilled in the art without departing from the spirit andscope of the example.

Example 40 Standard of Identity (SOI) and Non-Standard of Identity(Non-SOI) Dark and Milk Chocolate Formulations

[0573] Formulations of the compounds of the invention or combination ofcompounds derived by methods embodied in the invention can be preparedinto SOI and non-SOI dark and milk chocolates as a delivery vehicle forhuman and veterinary applications. Reference is made to copending U.S.application Ser. No. 08/709,406, filed Sep. 6, 1996, hereby incorporatedherein by reference. U.S. Ser. No. 08/709,406 relates to a method ofproducing cocoa butter and/or cocoa solids having conserved levels ofthe compounds of the invention from cocoa beans using a uniquecombination of processing steps. Briefly, the edible cocoa solidsobtained by this process conserves the natural occurrence of thecompounds of the invention in contrast to their levels found intraditionally processed cocoa, such that the ratio of the initial amountof the compounds of the invention found in the unprocessed bean to thatobtained after processing is less than or equal to 2. For simplicity,this cocoa solids material is designated herein as CP-cocoa solids. TheCP-cocoa solids are used as a powder or liquor to prepare SOI andnon-SOI chocolates, beverages, snacks, baked goods, and as an ingredientfor culinary applications.

[0574] The term “SOI chocolate” as used herein shall mean any chocolateused in food in the United States that is subject to a Standard ofIdentity established by the U.S. Food and Drug Administration under theFederal Food, Drug and Cosmetic Act. The U.S. definitions and standardsfor various types of chocolate are well established. The term “non-SOIchocolate” as used herein shall mean any nonstandardized chocolateswhich have compositions which fall outside the specified ranges of thestandardized chocolates.

[0575] Examples of nonstandardized chocolates result when the cocoabutter or milk fat are replaced partially or completely; or when thenutrative carbohydrate sweetener is replaced partially or completely; orflavors imitating milk, butter, cocoa powder, or chocolate are added orother additions or deletions in the formula are made outside the U.S.FDA Standards of Identity for chocolate or combinations thereof.

[0576] As a confection, chocolate can take the form of solid pieces ofchocolate, such as bars or novelty shapes, and can also be incorporatedas a component of other, more complex confections where chocolate isoptionally combined with any Flavor & Extract Manufacturers Association(FEMA) material, natural juices, spices, herbs and extracts categorizedas natural-flavoring substances; nature-identical substances; andartificial flavoring substances as defined by FEMA GRAS lists, FEMA andFDA lists, Council of Europe (CoE) lists, International Organization ofthe Flavor Industry (IOFI) adopted by the FAO/WHO Food StandardProgramme, Codex Alimentarius, and Food Chemicals Codex and generallycoats other foods such as caramel, nougat, fruit pieces, nuts, wafers orthe like. These foods are characterized as microbiologicallyshelf-stable at 65-85° F. under normal atmospheric conditions. Othercomplex confections result from surrounding with chocolate softinclusions such as cordial cherries or peanut butter. Other complexconfections result from coating ice cream or other frozen orrefrigerated desserts with chocolate. Generally, chocolate used to coator surround foods must be more fluid than chocolates used for plainchocolate solid bars or novelty shapes.

[0577] Additionally, chocolate can also be a low fat chocolatecomprising a fat and nonfat solids, having nutrative carbohydratesweetener(s), and an edible emulsifier. As to low fat chocolate,reference is made to U.S. Pat. Nos. 4,810,516, 4,701,337, 5,464,649,5,474,795, and WO 96/19923.

[0578] Dark chocolates derive their dark color from the amount ofchocolate liquor, or alkalized liquor or cocoa solids or alkalized cocoasolids used in any given formulation. However, the use of alkalizedcocoa solids or liquor would not be used in the dark chocolateformulations in the invention, since Example 27, Table 13 teaches theloss of the compounds of the invention due to the alkalization process.

[0579] Examples of formulations of SOI and non-SOI dark and milkchocolates are listed in Tables 16 and 17. In these formulations, theamount of the compounds of the invention present in CP-cocoa solids wascompared to the compounds of the invention present in commerciallyavailable cocoa solids.

[0580] The following describes the processing steps used in preparingthese chocolate formulations.

[0581] Process for Non-SOI Dark Chocolate

[0582] 1. Keep all mixers and refiners covered throughout process toavoid light.

[0583] 2. Batch all the ingredients excluding 40% of the free fat (cocoabutter and anhy. milk fat) maintaining temperature between 30-35° C.

[0584] 3. Refine to 20 microns.

[0585] 4. Dry conche for 1 hour at 35° C.

[0586] 5. Add full lechithin and 10% cocoa butter at the beginning ofthe wet conche cycle; wet conche for 1 hour.

[0587] 6. Add all remaining fat, standardize if necessary and mix for 1hour at 35° C.

[0588] 7. Temper, mould and package chocolate.

[0589] Process for SOI Dark Chocolate

[0590] 1. Batch all ingredients excluding milk fat at a temperature of60° C.

[0591] 2. Refine to 20 microns.

[0592] 3. Dry conche for 3.5 hours at 60° C.

[0593] 4. Add lecithin and milk fat and wet conche for 1 hour at 60° C.

[0594] 5. Standardize if necessary and mix for 1 hour at 35° C. Temper,mould and package chocolate.

[0595] Process for non-SOI Milk Chocolate

[0596] 1. Keep all mixers and refiners covered throughout process toavoid light.

[0597] 2. Batch sugar, whole milk powder, malted milk powder, and 66% ofthe cocoa butter, conche for 2 hours at 75° C.

[0598] 3. Cool batch to 35° C. and add cocoa powder, ethyl vanillin,chocolate liquor and 21% of cocoa butter, mix 20 minutes at 35° C.

[0599] 4. Refine to 20 microns.

[0600] 5. Add remainder of cocoa butter, dry conche for 1.5 hour at 35°C.

[0601] 6. Add anhy. milk fat and lecithin, wet conche for 1 hour at 35°C.

[0602] 7. Standardize, temper, mould and package the chocolate.

[0603] Process for SOI Milk Chocolate

[0604] 1. Batch all ingredients excluding 65% of cocoa butter and milkfat at a temperature of 60° C.

[0605] 2. Refine to 20 microns.

[0606] 3. Dry conche for 3.5 hours at 60° C.

[0607] 4. Add lecithin, 10% of cocoa butter and anhy. milk fat; wetconche for 1 hour at 60° C.

[0608] 5. Add remaining cocoa butter, standardize if necessary and mixfor 1 hour at 35° C.

[0609] 6. Temper, mould and package the chocolate.

[0610] The CP-cocoa solids and commercial chocolate liquors used in theformulations were analyzed for the pentamer and total level of monomersand compounds of the invention where n is 2 to 12 as described in Method2, Example 4 prior to incorporation in the formulations. These valueswere then used to calculate the expected levels in each chocolateformula as shown in Tables 16 and 17. In the cases for the non-SOI darkchocolate and non-SOI milk chocolate, their products were similarlyanalyzed for the pentamer, and the total level of monomers and thecompounds of the invention where n is 2 to 12. The results appear inTables 16 and 17.

[0611] The results from these formulation examples indicated that Soland non-SOI dark and milk chocolates formulated with CP-cocoa solidscontained approximately 6.5 times more expected pentamer, and 3.5 timesmore expected total levels in the SOI and non-SOI dark chocolates; andapproximately 4.5; 7.0 times more expected pentamer and 2.5; 3.5 timesmore expected total levels in the SOI and non-SOI milk chocolates,respectively.

[0612] Analyses of some of the chocolate products were not performedsince the difference between the expected levels of the compounds of theinvention present in finished chocolates prepared with CP-cocoa solidswere dramatically higher than those formulas prepared with commerciallyavailable cocoa solids. However, the effects of processing was evaluatedin the non-SOI dark and milk chocolate products. As shown in the tables,a 25-50% loss of the pentamer occurred, while slight differences intotal levels were observed. Without wishing to be bound by any theory,it is believed that these losses are due to heat and/or low chain fattyacids from the milk ingredient (e.g. acetic acid, propionic acid andbutyric acid) which can hydrolyze the oligomers (e.g. a trimer canhydrolyze to a monomer and dimer). Alternatively, time consumingprocessing steps can allow for oxidation or irreversible binding of thecompounds of the invention to protein sources within the formula. Thus,the invention comprehends altering methods of chocolate formulation andprocessing to address these effects to prevent or minimize these losses.

[0613] The skilled artisan will recognize many variations in theseexamples to cover a wide range of formulas, ingredients, processing, andmixtures to rationally adjust the naturally occurring levels of thecompounds of the invention for a variety of chocolate applications.TABLE 16 Dark Chocolate Formulas Prepared with non-Alkalized CocoaIngredients Non-SOI SOI Dark Chocolate SOI Dark Chocolate Dark ChocolateUsing Using Using Commerical CP-cocoa solids CP-Cocoa Solids CocoaFormulation: Formulation: Formulation: 41.49% Sugar 41.49% sugar 41.49%sugar    3% whole milk    3% whole milk    3% whole milk        powder       powder        powder   26% CP-cocoa solids 52.65% CP-liquor52.65% corn, liquor  4.5% corn, liquor  2.35% anhy. milk fat  2.35%anhy. milk        fat 21.75% cocoa butter  0.01% vanillin  0.01%vanillin  2.75% anhy. milk fat  0.5% lecithin  0.5% lecithin  0.01%vanillin  0.5% lecithin Total fat: 31% Total fat: 31% Total fat: 31%Particle size: 20 microns Particle size: 20 microns Particle size: 20microns Expected Levels of pentamer and total oligomeric procyanidins(monomers and n = 2-12; units of ug/g) Pentamer: 1205 Pentamer: 1300Pentamer: 185 Total: 13748 Total: 14646 Total: 3948 Actual Levels ofpentamer and total oligomeric procyandins (monomers and n = 2-12; unitsof ug/g) Pentamer: 561 Not performed Not performed Total: 14097

[0614] TABLE 17 Milk Chocolate Formulas Prepared with non-AlkalizedCocoa Ingredients SOI Milk Chocolate SOI Milk Chocolate Non-SOI MilkChocolate Using CP-Cocoa Using Commerical Using CP-cocoa solids SolidsCocoa Solids Formulation: Formulation: Formulation: 46.9965% Sugar46.9965% sugar 46.9965% sugar   15.5% whole milk   15.5% whole milk  15.5% whole milk         powder         powder         powder   4.5%CP-cocoa   13.9% CP-liquor   13.9% corn, liquor         solids   5.5%corn. liquor   1.6% anhy. milk   1.60% anhy. milk         fat        fat   21.4% cocoa butter  0.0035% vanillin  0.0035% vanillin   1.6%anhy. milk fat   0.5% lecithin   0.5% lecithin  0.035% vanillin   17.5%cocoa butter   17.5% cocoa butter   0.5% lecithin   4.0% malted milk  4.0% malted milk         powder         powder   4.0% malted milk       powder Total fat: 31.75% Total fat: 31.75% Total fat: 31.75%Particle size: 20 microns Particle size: 20 Particle size: 20 micronsmicrons Expected Levels of pentamer and total oligomeric procyanidins(monomers and n = 2-12; units of ug/g) Pentamer: 225 Pentamer: 343Pentamer: 49 Total: 2734 Total: 3867 Total: 1042 Actual Levels ofpentamer and total oligomeric procyandins (monomers and n = 2-12; unitsof ug/g) Pentamer: 163 Not performed Not performed Total: 2399

Example 41 Hydrolysis of Procyanidin Oligomers

[0615] Example 14, Method D describes the preparation normal phase HPLCprocedure to purify the compounds of the invention. The oligomers areobtained as fractions dissolved in mobile phase. Solvent is then removedby standard vacuum distillation (20-29 in. Hg: 40° C.) on a Rotovapapparatus. It was observed that losses of a particular oligomer occurredwith increases in smaller oligomers when the vacuum distillationresidence time was prolonged or temperatures >40° C. were used.

[0616] The losses of a particular oligomer with accompanying increasesin smaller oligomers was attributed to a time-temperature acidhydrolysis from residual acetic acid present in the mobile phase solventmixture. This observation was confirmed by the following experimentwhere 100 mg of hexamer was dissolved in 50 mL of the mobile phasecontaining methylene chloride, acetic acid, water, and methanol (seeExample 14, Method D for solvent proportions) and subjected to atime-temperature dependent distillation. At specific times, an aliquotwas removed for analytical normal phase HPLC analysis as described inExample 4, Method 2. The results are illustrated in FIGS. 64 and 65,where hexamer levels decreased in a time-temperature dependent fashion.FIG. 65 illustrates the appearance of one of the hydrolysis products(Trimer) in a time-temperature dependent fashion. Monomer and otheroligomers (dimer to pentamer) also appeared in a time-temperaturedependent fashion.

[0617] These results indicated that extreme care and caution must betaken during the handling of the inventive polymeric compounds.

[0618] The results provided above, together with that found in Examples5, 15, 18, 19, 20 and 29, demonstrate that the method described abovecan be used to complement other methods embodied in the invention toidentify any given oligomer of the invention.

[0619] For instance, the complete hydrolysis of any given oligomer whichyields exclusively (+)-catechin or (−)-epicatechin eliminates many“mixed” monomer-based oligomer structure possibilities and reduces thestereochemical linkage possibilities characteristic for each monomercomprising any given oligomer.

[0620] Further, the complete hydrolysis of any given oligomer whichyields both (+)-catechin and (−)-epidatechin in specific proportionsprovides the skilled artisan with information on the monomer compositionof any given oligomer, and hence, the stereochemical linkagepossibilities characteristic for each monomer comprising the oligomer.

[0621] The skilled artisan would recognize the fact that acid catalyzedepimerization of individual monomers can occur and suitable controlexperiments and nonvigorous hydrolysis conditions should be taken intoaccount (e.g., the use of an organic acids, such as acetic acid, in lieuof concentrated HCl, HNO₃, etc).

[0622] Having thus described in detail the preferred embodiments of thepresent invention, it is to be understood that the invention defined bythe appended claims is not to be limited by particular details set forthin the above descriptions as many apparent variations thereof arepossible without departing from the spirit or scope of the presentinvention.

What is claimed is:
 1. A polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 3 to 18, such that there is at least oneterminal monomeric unit A, and a plurality of additional monomericunits; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar; bondingbetween adjacent monomers takes place at positions selected from thegroup consisting of 4, 6 and 8; a bond for an additional monomeric unitin position 4 has alpha or beta stereochemistry; X, Y and Z are selectedfrom the group consisting of monomeric unit A, hydrogen, and a sugar,with the provisos that as to the at least one terminal monomeric unit,bonding of the additional monomeric unit thereto is at position 4 andoptionally Y=Z=hydrogen; the sugar is optionally substituted with aphenolic moiety, and pharmaceutically acceptable salts, derivativesthereof, and oxidation products thereof.
 2. The compound of claim 1wherein n is
 5. 3. The compound of claim 1 wherein the sugar is selectedfrom the group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 4. The compound of claim 1 which is isolated from a naturalsource.
 5. The compound of claim 4 wherein the natural source is aTheobroma or Herrania species or inter- or intra-species specificcrosses thereof.
 6. The compound of claim 1 which is substantially pure.7. The compound of claim 1 which is purified to apparent homogeneity. 8.The compound of claim 1 wherein the phenolic moiety is selected from thegroup consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 9. An antineoplastic compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 3 to 18, such that there is at least oneterminal monomeric unit A, and a plurality of additional monomericunits; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or 3-(β)—O-sugar; bondingbetween adjacent monomers takes place at positions selected from thegroup consisting of 4, 6 and 8; a bond of an additional monomeric unitin position 4 has alpha or beta stereochemistry; X, Y and Z are selectedfrom the group consisting of monomeric unit A, hydrogen, and a sugar,with the provisos that as to the at least one terminal monomeric unit,bonding of the additional monomeric unit thereto is at position 4 andoptionally Y=Z=hydrogen; the sugar is optionally substituted with aphenolic moiety; pharmaceutically acceptable salts, derivatives thereof,and oxidation products thereof; and a pharmaceutically acceptablecarrier or excipient.
 10. The composition of claim 9 wherein n is 5 to12.
 11. The composition of claim 9 wherein the sugar is selected fromthe group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 12. The composition of claim 9 wherein said polymericcompound is substantially pure.
 13. The composition of claim 9 whereinthe phenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.14. The composition of claim 9 wherein n is
 5. 15. A method for treatinga subject in need of treatment with an antineoplastic agent comprisingadministering to the subject an antineoplastic composition as claimed inclaim
 9. 16. The method of claim 15, further comprising administering atleast one additional antineoplastic agent.
 17. A kit for a compositionof claim 9 comprising the compound and the carrier or diluent separatelypackaged, and optionally instructions for admixture or administration.18. A composition as claimed in claim 9 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 19. Anantioxidant composition comprising a polymeric compound of the formulaAn, wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 20. Thecomposition of claim 19 wherein n is 2 to
 10. 21. The composition ofclaim 19 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 22. The compositionof claim 19 wherein said polymeric compound is substantially pure. 23.The composition of claim 19 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 24. The composition of claim 19wherein n is 2 to
 12. 25. A method for treating a subject in need oftreatment with an antioxidant agent comprising administering to thesubject an antioxidant composition as claimed in claim
 19. 26. Themethod of claim 25, further comprising administering at least oneadditional antioxidant agent.
 27. A composition as claimed in claim 19wherein the composition is selected from the group consisting of atablet, capsule, Standard of Identity chocolate and non-Standard ofIdentity chocolate.
 28. A kit for a composition of claim 19 comprisingthe compound and the carrier or diluent separately packaged, andoptionally instructions for admixture or administration.
 29. A methodfor preserving or protecting a desired item from oxidation comprisingcontacting the item with a composition as claimed in claim
 19. 30. Anantimicrobial composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 31. Thecomposition of claim 30 wherein n is 2 to
 10. 32. The composition ofclaim 30 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 33. The compositionof claim 30 wherein said polymeric compound is substantially pure. 34.The composition of claim 30 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 35. The composition of claim 30wherein n is selected from the group consisting of 2, 4, 5, 6, 8 and 10.36. A composition as claimed in claim 30 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 37. A methodfor treating a subject in need of treatment with an antimicrobial agentcomprising administering to the subject an antimicrobial composition asclaimed in claim
 30. 38. The method of claim 37, further comprisingadministering at least one additional antimicrobial agent.
 39. A kit fora composition of claim 30 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 40. A cyclo-oxygenase and/or lipoxygenase modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 41. Thecomposition of claim 40 wherein n is 2 to
 10. 42. The composition ofclaim 40 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 43. The compositionof claim 40 wherein said polymeric compound is substantially pure. 44.The composition of claim 40 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 45. The composition of claim 40wherein n is 2 to
 5. 46. A composition as claimed in claim 40 whereinthe composition is selected from the group consisting of a tablet,capsule, Standard of Identity chocolate and non-Standard of Identitychocolate.
 47. A method for treating a subject in need of treatment witha cyclo-oxygenase and/or lipoxygenase modulating agent comprisingadministering to the subject a composition as claimed in claim
 40. 48.The method of claim 47, further comprising administering at least oneadditional cyclo-oxygenase and/or lipoxygenase modulating agent.
 49. Akit for a composition of claim 40 comprising the compound and thecarrier or diluent separately packaged, and optionally instructions foradmixture or administration.
 50. An NO or NO-synthase modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety via an esterbond; pharmaceutically acceptable salts, derivatives thereof, andoxidation products thereof; and a pharmaceutically acceptable carrier orexcipient.
 51. The composition of claim 50 wherein n is 2 to
 10. 52. Thecomposition of claim 50 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 53.The composition of claim 50 wherein said polymeric compound issubstantially pure.
 54. The composition of claim 50 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 55. Acomposition as claimed in claim 50 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 56. A method fortreating a subject in need of treatment with a NO-modulating agentcomprising administering to the subject an NO or NO-synthase modulatingcomposition as claimed in claim
 50. 57. The method of claim 56, furthercomprising administering at least one additional NO or NO-synthasemodulating agent.
 58. A kit for a composition of claim 50 comprising thecompound and the carrier or diluent separately packaged, and optionallyinstructions for admixture or administration.
 59. A method of treating amammal in need of treatment with an NO or NO synthase agent comprisingadministering to the mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 60. A method ofreducing No affected hypercholesterolemia in a mammal comprisingadministering to said mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 61. The methodof claim 60 wherein n is 2 to
 10. 62. A method for the induction of iNOSin mammalian monocyte and/or macrophages comprising contacting saidmonocyte and/or macrophages with a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 63. A method ofstimulating mammalian monocyte and/or macrophage NO productioncomprising administering to said mammal a composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 64. The methodof claim 63 wherein n is 2 to
 10. 65. An in vivo glucose-modulatingcomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 66. Thecomposition of claim 65 wherein n is 2 to
 10. 67. The composition ofclaim 65 wherein the sugar is selected from the group consisting ofglucose, galactose, xylose, rhamnose and arabinose.
 68. The compositionof claim 65 wherein said polymeric compound is substantially pure. 69.The composition of claim 65 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 70. A composition as claimed in claim65 wherein the composition is selected from the group consisting of atablet, capsule, Standard of Identity chocolate and non-Standard ofIdentity chocolate.
 71. A method for treating a subject in need oftreatment with a glucose-modulating agent comprising administering tothe subject a glucose-modulating agent as claimed in claim
 65. 72. Themethod of claim 71, further comprising administering at least oneadditional glucose-modulating agent.
 73. A kit for a composition ofclaim 65 comprising the compound and the carrier or diluent separatelypackaged, and optionally instructions for admixture or administration.74. A topoisomerase II-inhibiting composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 75. Thecomposition of claim 74 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 76.The composition of claim 74 wherein said polymeric compound issubstantially pure.
 77. The composition of claim 74 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamin,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 78. Acomposition as claimed in claim 74 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 79. A method forinhibiting topoisomerase II which comprises contacting topoisomerase IIwith a composition as claimed in claim
 74. 80. A kit for a compositionof claim 74 comprising the compound and the carrier or diluentseparately packaged, and optionally instructions for admixture oradministration.
 81. A non-steroidal antiinflammatory compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond ofthe additional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of an additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety, andpharmaceutically acceptable salts, derivatives thereof, and oxidationproducts thereof; and a pharmaceutically acceptable carrier orexcipient.
 82. The composition of claim 81 wherein the sugar is selectedfrom the group consisting of glucose, galactose, xylose, rhamnose andarabinose.
 83. The composition of claim 81 wherein said polymericcompound is substantially pure.
 84. The composition of claim 81 whereinthe phenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.85. A composition as claimed in claim 81 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 86. A methodfor treating a subject in need of treatment with a non-steroidalantiinflammatory agent comprising administering to the subject acomposition as claimed in claim
 81. 87. The method of claim 86, furthercomprising administering at least one additional non-steroidalantiinflammatory agent.
 88. A kit for a composition of claim 81comprising the compound and the carrier or diluent separately packaged,and optionally instructions for admixture or administration.
 89. Ananti-gingivitis or anti-periodontitis composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 90. Thecomposition of claim 89 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 91.The composition of claim 89 wherein said polymeric compound issubstantially pure.
 92. The composition of claim 89 wherein the phenolicmoiety is selected from the group consisting of caffeic, cinnamic,coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.
 93. Acomposition as claimed in claim 89 wherein the composition is selectedfrom the group consisting of a tablet, capsule, Standard of Identitychocolate and non-Standard of Identity chocolate.
 94. A kit for acomposition of claim 89 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 95. A method of reducing periodontal diseaseprogression in a mammal comprising administering to said mammal with acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 96. The methodof claim 95 wherein n is 2 to
 10. 97. A method of treatment ofperiodontal disease comprising administering to a mammal in need of suchtreatment a composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 98. A plateletaggregation modulating composition comprising a polymeric compound ofthe formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 99. Thecomposition of claim 98 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 100.The composition of claim 98 wherein said polymeric compound issubstantially pure.
 101. The composition of claim 98 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.102. A composition as claimed in claim 98 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 103. A kitfor a composition of claim 98 comprising the compound and the carrier ordiluent separately packaged, and optionally instructions for admixtureor administration.
 104. A method of modulating platelet aggregationadministering to a mammal in need of such treatment with a compound ofclaim
 98. 105. An apoptosis modulating composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 106. Thecomposition of claim 105 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 107.The composition of claim 105 wherein said polymeric compound issubstantially pure.
 108. The composition of claim 105 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.109. A composition as claimed in claim 105 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 110. A kitfor a composition of claim 105 comprising the compound and the carrieror diluent separately packaged, and optionally instructions foradmixture or administration.
 111. A method of modulating apoptosiscomprising contacting COX-2 expressing cancer cells with a compositioncomprising a polymeric compound of the formula A_(n), wherein A is amonomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 112. The methodof claim 111 wherein n is
 5. 113. A method of inhibiting oxidativedamage to DNA comprising contacting DNA with a composition comprising apolymeric compound of the formula A_(n), wherein A is a monomer of theformula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 114. Thecomposition of claim 113 wherein the sugar is selected from the groupconsisting of glucose, galactose, xylose, rhamnose and arabinose. 115.The composition of claim 113 wherein said polymeric compound issubstantially pure.
 116. The composition of claim 113 wherein thephenolic moiety is selected from the group consisting of caffeic,cinnamic, coumaric, ferulic, gallic, hydroxybenzoic and sinapic acids.117. A composition as claimed in claim 113 wherein the composition isselected from the group consisting of a tablet, capsule, Standard ofIdentity chocolate and non-Standard of Identity chocolate.
 118. A kitfor a composition of claim 113 comprising the compound and the carrieror diluent separately packaged, and optionally instructions foradmixture or administration.
 119. A composition for treating, preventingor reducing atherosclerosis or restenosis comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or O-sugar;bonding between adjacent monomers takes place at positions selected fromthe group consisting of 4, 6 and 8; a bond of an additional monomericunit in position 4 has alpha or beta stereochemistry; X, Y and Z areselected from the group consisting of monomeric unit A, hydrogen, and asugar, with the provisos that as to the at least one terminal monomericunit, bonding of the additional monomeric unit thereto is at position 4and optionally Y=Z=hydrogen; the sugar is optionally substituted with aphenolic moiety; pharmaceutically acceptable salts, derivatives thereof,and oxidation products thereof; and a pharmaceutically acceptablecarrier or excipient.
 120. The composition of claim 119 wherein thesugar is selected from the group consisting of glucose, galactose,xylose, rhamnose and arabinose.
 121. The composition of claim 119wherein said polymeric compound is substantially pure.
 122. Thecomposition of claim 119 wherein the phenolic moiety is selected fromthe group consisting of caffeic, cinnamic, coumaric, ferulic, gallic,hydroxybenzoic and sinapic acids.
 123. A composition as claimed in claim119 wherein the composition is selected from the group consisting of atablet, capsule, Standard of Identity chocolate and non-Standard ofIdentity chocolate.
 124. A method of treating, preventing or reducingatherosclerosis or restenosis in a mammal comprising administering tosaid mammal a composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 125. The methodof claim 124 wherein n is 2 to
 10. 126. A method of inhibiting theoxidation of LDL in a mammal comprising administering to said mammal acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 127. The methodof claim 126 wherein n is 2 to
 12. 128. A method of modulatingthrombosis in a mammal comprising administering to said mammal acompound of claim 1 and a suitable carrier or diluent.
 129. The methodof claim 128 wherein n is 2 to
 12. 130. A method of modulatingcyclooxygenase-1 (COX-1) for the treatment for inflammatory boweldisease comprising administering to a mammal in need of such treatment acomposition comprising a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3(β)—O-sugar; bonding between adjacent monomers takes place at positionsselected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 131. A method ofinhibiting bacterial growth in a mammal comprising administering to saidmammal a composition comprising a polymeric compound of the formulaA_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 132. The methodof claim 131 wherein n is 2, 4, 5, 6, 8 and
 10. 133. A method ofreducing hypertension in a mammal in need of such treatment comprisingadministering to said mammal a composition comprising a polymericcompound of the formula A_(n), wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond of anadditional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety; pharmaceuticallyacceptable salts, derivatives thereof, and oxidation products thereof;and a pharmaceutically acceptable carrier or excipient.
 134. She methodof claim 133 wherein n is 2 to
 10. 135. A method for treating cancer ina mammal in need of such treatment, comprising administering to themammal in an amount sufficient to effect said treatment of at least onecompound in claim
 1. 136. The method of claim 135 wherein said cancer isprostate cancer.
 137. The method of claim 135 wherein said cancer isrenal cancer.
 138. The method of claim 135 wherein said cancer is coloncancer.
 139. The method of claim 135 wherein said cancer is breastcancer.
 140. The method of claim 135 wherein the cancer is felineleukemia.
 141. The method of claim 135 wherein the cancer is cervicalcancer.
 142. The method of claim 135 wherein the cancer is T-cellleukemia.
 143. A method of arresting cancer cell growth in a mammalcomprising administering to said mammal a compound of claim 1 and asuitable carrier or diluent.
 144. The method of claim 143 wherein n is5.
 145. A carrier or vehicle for a pharmaceutical comprising a cocoaextract.
 146. A substantially pure polyphenol from Theobroma or Herraniaspecies or inter- or intra-species specific crosses thereof comprisingpolyphenols comprising oligomers 3 through
 18. 147. A compound of claim1 having a structure giving an NMR spectra as set forth in FIGS. 48A-D.148. A compound of claim 1 having a structure giving an NMR spectra asset forth in FIGS. 49A-D.
 149. A method for enhancing the concentrationlevels and distribution of cocoa procyanidins in cocoa beans bymanipulating fermentation conditions.
 150. The compound as claimed inclaim 1 wherein the compound is a trimer of formula [EC-(4β→8)]₂-EC.151. The compound as claimed in claim 1 wherein the compound is atetramer of formula [EC-(4β→8)]₃-EC.
 152. The compound as claimed inclaim 1 wherein the compound is a pentamer of formula [EC-(4β→8)]₄-EC,wherein a 3-position of the terminal monomeric unit of the pentamer isoptionally derivatized with a gallate and/or β-D-glucose.
 153. Thecompound as claimed in claim 1 wherein the compound is a hexamer offormula [EC-(4β→8)]₅-EC.
 154. The compound as claimed in claim 1 whereinthe compound is a heptamer of formula [EC-(4β→8)]₆-EC.
 155. The compoundas claimed in claim 1 wherein the compound is an octamer of formula[EC-(4β→8)]₇-EC.
 156. The compound as claimed in claim 1 wherein thecompound is a nonamer of formula [EC-(4β→8)]₈-EC.
 157. The compound asclaimed in claim 1 wherein the compound is a decamer of formula[EC-(4β→8)]₉-EC.
 158. The compound as claimed in claim 1 wherein thecompound is an undecamer of formula [EC-(4β→8)]₁₀-EC.
 159. The compoundas claimed in claim 1 wherein the compound is a dodecamer of formula[EC-(4β→8)]₁₁-EC.
 160. The composition as claimed in claim 9 wherein thecompound is selected from the group consisting of a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, a decamer of formula[EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC, and adodecamer of formula [EC-(4β→8)]₁₁-EC.
 161. The composition as claimedin claim 19 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(45-8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 162. The composition as claimed in claim 30 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, and a decamer of formula [EC-(4β→8)]₉-EC.
 163. Thecomposition as claimed in claim 40 wherein the compound is selected fromthe group consisting of a dimer of formula EC-(4β→8)-EC, a trimer offormula [EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, apentamer of formula [EC-(4β→8)]₄-EC, a hexamer of formula[EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, an octamer offormula [EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4β→8)]₈-EC, and adecamer of formula [EC-(4β→8)]₉-EC.
 164. The composition as claimed inclaim 50 wherein the compound is selected from the group consisting of adimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, atetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 165. The composition as claimed in claim 65 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, and a decamer of formula [EC-(4β→8)]₉-EC.
 166. Thecomposition as claimed in claim 74 wherein the compound is selected fromthe group consisting of a dimer of formula EC-(4β→8)-EC, a trimer offormula [EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, apentamer of formula [EC-(4β→8)]₄-EC, a hexamer of formula[EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, an octamer offormula [EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4β→8)]₈-EC, a decamerof formula [EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC,and a dodecamer of formula [EC-(4β→8)]₁₁-EC.
 167. The composition asclaimed in claim 81 wherein the compound is selected from the groupconsisting of a dimer of formula EC-(4β→8)-EC, a trimer of formula[EC-(4β→8)]₂-EC, a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer offormula [EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, aheptamer of formula [EC-(4β→8)]₆-EC, an octamer of formula[EC-(4β→8)]₇-EC, a nonamer of formula [EC-(4β→8)]₈-EC, a decamer offormula [EC-(4β→8)]₉-EC, an undecamer of formula [EC-(4β→8)]₁₀-EC, and adodecamer of formula [EC-(4β→8)]₁₁-EC.
 168. The composition as claimedin claim 89 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 169. The composition as claimed in claim 98 wherein thecompound is selected from the group consisting of a dimer of formulaEC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC, a tetramer of formula[EC-(4β→8)]₃-EC, a pentamer of formula [EC-(4β→8)]₄-EC, a hexamer offormula [EC-(4β→8)]₅-EC, a heptamer of formula [EC-(4β→8)]₆-EC, anoctamer of formula [EC-(4β→8)]₇-EC, a nonamer of formula[EC-(4β→8)]₈-EC, a decamer of formula [EC-(4β→8)]₉-EC, an undecamer offormula [EC-(4β→8)]₁₀-EC, and a dodecamer of formula [EC-(4β→8)]₁₁-EC.170. The composition as claimed in claim 105 wherein the compound is apentamer of formula [EC-(4β→8)]₄-EC.
 171. The compositition as claimedin claim 119 wherein the compound is selected from the group consistingof a dimer of formula EC-(4β→8)-EC, a trimer of formula [EC-(4β→8)]₂-EC,a tetramer of formula [EC-(4β→8)]₃-EC, a pentamer of formula[EC-(4β→8)]₄-EC, a hexamer of formula [EC-(4β→8)]₅-EC, a heptamer offormula [EC-(4β→8)]₆-EC, an octamer of formula [EC-(4β→8)]₇-EC, anonamer of formula [EC-(4β→8)]₈-EC, and a decamer of formula[EC-(4β→8)]₉-EC.
 172. A method for the identification of at least onegene induced or repressed by a polymeric compound of the formula A_(n),wherein A is a monomer of the formula:

wherein n is an integer from 2 to 18, such that there is at least oneterminal monomeric unit A, and one or a plurality of additionalmonomeric units; R is 3-(α)—OH, 3-(β)—OH, 3-(α)—O-sugar, or3-(β)—O-sugar; bonding between adjacent monomers takes place atpositions selected from the group consisting of 4, 6 and 8; a bond foran additional monomeric unit in position 4 has alpha or betastereochemistry; X, Y and Z are selected from the group consisting ofmonomeric unit A, hydrogen, and a sugar, with the provisos that as tothe at least one terminal monomeric unit, bonding of the additionalmonomeric unit thereto is at position 4 and optionally Y=Z=hydrogen; thesugar is optionally substituted with a phenolic moiety, andpharmaceutically acceptable salts, derivatives thereof, oxidationproducts thereof, or combinations thereof, said method comprisingcontacting said at least one gene or a gene product thereof with thepolymeric compound using a gene expression assay.
 173. The method asclaimed in claim 172 wherein said gene expression assay is selected fromthe group consisting of Differential Display, sequencing of cDNAlibraries, Serial Analysis of Gene Expression, and expression monitoringby hybridization to high density oligonucleotide arrays.
 174. Thecompound as claimed in claim 1, wherein adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 175. The composition as claimed in claim 9, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 176. The composition asclaimed in claim 19, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 177. The composition as claimed in claim 30,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 178. The composition as claimed in claim 40, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 179. The composition asclaimed in claim 50, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 180. The composition as claimed in claim 65,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 181. The composition as claimed in claim 74, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 182. The composition asclaimed in claim 81, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 183. The composition as claimed in claim 89,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 184. The composition as claimed in claim 98, wherein thecompound further comprises adjacent monomers bind at position 4 by (4→6)or (4→8); and each of X, Y and Z is H, a sugar or an adjacent monomer,with the provisos that if X and Y are adjacent monomers, Z is H or sugarand if X and Z are adjacent monomers, Y is H or sugar, and that as to atleast one of the two terminal monomers, bonding of the adjacent monomeris at position 4 and optionally, Y=Z=hydrogen.
 185. The composition asclaimed in claim 105, wherein the compound further comprises adjacentmonomers bind at position 4 by (4→6) or (4→8); and each of X, Y and Z isH, a sugar or an adjacent monomer, with the provisos that if X and Y areadjacent monomers, Z is H or sugar and if X and Z are adjacent monomers,Y is H or sugar, and that as to at least one of the two terminalmonomers, bonding of the adjacent monomer is at position 4 andoptionally, Y=Z=hydrogen.
 186. The composition as claimed in claim 119,wherein the compound further comprises adjacent monomers bind atposition 4 by (4→6) or (4→8); and each of X, Y and Z is H, a sugar or anadjacent monomer, with the provisos that if X and Y are adjacentmonomers, Z is H or sugar and if X and Z are adjacent monomers, Y is Hor sugar, and that as to at least one of the two terminal monomers,bonding of the adjacent monomer is at position 4 and optionally,Y=Z=hydrogen.
 187. The compound as claimed in claim 1 wherein thecompound is a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]-EC.
 188. The compound asclaimed in claim 1 wherein the compound is a tetramer selected from thegroup consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C.
 189. The compound as claimed in claim 1wherein the compound is a pentamer selected from the group consisting of[EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C.
 190. The compound as claimed in claim 1wherein the compound is a hexamer selected from the group consisting of[EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, wherein the 3-position of a hexamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 191. The compound as claimed in claim 1 wherein thecompound is a heptamer selected from the group consisting of[EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, wherein the 3-position of the heptamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 192. The compound as claimed in claim 1 wherein thecompound is an octamer selected from the group consisting of[EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, wherein the 3-position of an octamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 193. The compound as claimed in claim 1 wherein thecompound is a nonamer selected from the group consisting of[EC-(4β→8)]8-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, wherein the 3-position of a nonamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 194. The compound as claimed in claim 1 wherein thecompound is a decamer selected from the group consisting of[EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,and [EC-(4β→8)]₈-EC-(4β→6)-C, wherein the 3-position of a decamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 195. The compound as claimed in claim 1 wherein thecompound is an undecamer selected from the group consisting of[EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,and [EC-(4β→8)]₉-EC-(4β→6)-C, wherein the 3-position of an undecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 196. The compound as claimed in claim 1 wherein thecompound is a dodecamer selected from the group consisting of[EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C,and [EC-(4β→8)]₁₀-EC-(4β→6)-C, wherein the 3-position of a dodecamerterminal monomeric unit is optionally derivatized with a gallate or aβ-D-glucose.
 197. The composition as claimed in claim 9 wherein thecompound is selected from the group consisting of a pentamer selectedfrom the group consisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamerselected from the group consisting of [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the group consistingof [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the groupconsisting of [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC,[EC-(4β→8)]₆-EC-(4β→8)-C, and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamerselected from the group consisting of [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)-EC-(4β→6)-C, a decamer selected from the group consisting of[EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,and [EC-(4β→8)]₈-EC-(4β→6)-C, an undecamer selected from the groupconsisting of [EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC,[EC-(4β→8)]₉-EC-(4β→8)-C, and [EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamerselected from the group consisting of [EC-(4β→8)]₁₁-EC,[EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C, and[EC-(4β→8)]₁₀-EC-(4β→6)-C.
 198. The composition as claimed in claim 19wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, (EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 199. Thecomposition as claimed in claim 30 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C.
 200. The composition as claimed in claim 40wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 201. Thecomposition as claimed in claim 50 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,(EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C.
 202. The composition as claimed in claim 65wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.
 203. Thecomposition as claimed in claim 74 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C, an undecamer selected from the groupconsisting of [EC-(4β→8)]₁₀-EC, [EC-(4β→8)]₉-EC-(4β→6)-EC,[EC-(4β→8)]₉-EC-(4β→8)-C, and [EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamerselected from the group consisting of [EC-(4β→8)]₁₁-EC,[EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C, and[EC-(4β→8)]₁₀-EC-(4β→6)-C.
 204. The composition as claimed in claim 81wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C, an undecamerselected from the group consisting of [EC-(4β→8)]₁₀-EC,[EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C, and[EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamer selected from the groupconsisting of [EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC,[EC-(4β→8)]₁₀-EC-(4β→8)-C, and [EC-(4β→8)]₁₀-EC-(4β→6)-C.
 205. Thecomposition as claimed in claim 89 wherein the compound is selected fromthe group consisting of a trimer selected from the group consisting of[EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramer selectedfrom the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and[EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the group consistingof [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-Cand [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the groupconsisting of [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→8)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamerselected from the group consisting of [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the group consistingof [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C,and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the groupconsisting of [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C, and [EC-(4β→8)]₇-EC-(4β→6)-C, and a decamerselected from the group consisting of [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C, and[EC-(4β→8)]₈-EC-(4β→6)-C.
 206. The composition as claimed in claim 98wherein the compound is selected from the group consisting of a trimerselected from the group consisting of [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-Cand [EC-(4β→6)]₂-EC, a tetramer selected from the group consisting of[EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C and [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamerselected from the group consisting of [EC-(4β→8)]₄-EC,[EC-(4β→8)]₃-EC-(4β→6)-EC, [EC-(4β→8)]₃-EC-(4β→8)-C and[EC-(4β→8)]₃-EC-(4β→6)-C, a hexamer selected from the group consistingof [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C,and [EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the groupconsisting of [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC,[EC-(4β→8)]₅-EC-(4β→8)-C, and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamerselected from the group consisting of [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→8)-C, and[EC-(4β→8)]₆-EC-(4β→6)-C, a nonamer selected from the group consistingof [EC-(4β→8)]₈-EC, [EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C,and [EC-(4β→8)]₇-EC-(4β→6)-C, a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C, an undecamerselected from the group consisting of [EC-(4β→8)]₁₀-EC,[EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C, and[EC-(4β→8)]₉-EC-(4β→6)-C, and a dodecamer selected from the groupconsisting of [EC-(4β→8)]₁₁-EC, [EC-(4β→8)]₁₀-EC-(4β→6)-EC,[EC-(4β→8)]₁₀-EC-(4β→8)-C, and [EC-(4β→8)]₁₀-EC-(4β→6)-C.
 207. Thecomposition as claimed in claim 105 wherein the compound is selectedfrom the group consisting of a pentamer selected from the groupconsisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C.
 208. Thecomposition as claimed in claim 119 wherein the compound is selectedfrom the group consisting of a trimer selected from the group consistingof [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C and [EC-(4β→6)]₂-EC, a tetramerselected from the group consisting of [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-Cand [EC-(4β→8)]₂-EC-(4β→6)-C, a pentamer selected from the groupconsisting of [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C and [EC-(4β→8)]₃-EC-(4β→6)-C, a hexamerselected from the group consisting of [EC-(4β→8)]₅-EC,[EC-(4β→8)]₄-EC-(4β→6)-EC, [EC-(4β→8)]₄-EC-(4β→8)-C, and[EC-(4β→8)]₄-EC-(4β→6)-C, a heptamer selected from the group consistingof [EC-(4β→8)]₆-EC, [EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C,and [EC-(4β→8)]₅-EC-(4β→6)-C, an octamer selected from the groupconsisting of [EC-(4β→8)]₇-EC, [EC-(4β→8)]₆-EC-(4β→6)-EC,[EC-(4β→8)]₆-EC-(4β→8)-C, and [EC-(4β→8)]₆-EC-(4β→6)-C, a nonamerselected from the group consisting of [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC, [EC-(4β→8)]₇-EC-(4β→8)-C, and[EC-(4β→8)]₇-EC-(4β→6)-C, and a decamer selected from the groupconsisting of [EC-(4β→8)]₉-EC, [EC-(4β→8)]₈-EC-(4β→6)-EC,[EC-(4β→8)]₈-EC-(4β→8)-C, and [EC-(4β→8)]₈-EC-(4β→6)-C.